Objectives. Recently, the field of tissue engineering has made numerous advances towards achieving artificial tendon substitutes with excellent mechanical and histological properties, and has had some promising experimental results. The purpose of this systematic review is to assess the efficacy of tissue engineering in the treatment of tendon injuries. Methods. We searched MEDLINE, Embase, and the Cochrane Library for the time period 1999 to 2016 for trials investigating tissue engineering used to improve tendon healing in animal models. The studies were screened for inclusion based on randomization, controls, and reported measurable outcomes. The RevMan software package was used for the meta-analysis. Results. A total of 388 references were retrieved and 35 studies were included in this systematic review. The different biomaterials developed were analyzed and we found that they improve the biomechanical and histological characteristics of the repaired tendon. At meta-analysis, despite a high heterogeneity, it revealed a statistically significant effect in favour of the maximum load, the maximum stress, and the Young’s modulus between experimental and control groups. In the forest plot, the diamond was on the right side of the vertical line and did not intersect with the line, favouring experimental groups. Conclusions. This review of the literature demonstrates the heterogeneity in the tendon tissue engineering literature. Several biomaterials have been developed and have been shown to enhance tendon healing and regeneration with improved outcomes. Cite this article: D. González-Quevedo, I. Martínez-Medina, A. Campos, F. Campos, V. Carriel. Tissue engineering strategies for the treatment of tendon injuries: a systematic review and meta-analysis of animal models. Bone Joint Res 2018;7:318–324. DOI: 10.1302/2046-3758.74.BJR-2017-0326

When transferring tissue regenerative strategies involving skeletal stem cells to human application, consideration needs to be given to factors that may affect the function of the cells that are transferred. Local anaesthetics are frequently used during surgical procedures, either administered directly into the operative site or infiltrated subcutaneously around the wound. The aim of this study was to investigate the effects of commonly used local anaesthetics on the morphology, function and survival of human adult skeletal stem cells.

Cells from three patients who were undergoing elective hip replacement were harvested and incubated for two hours with 1% lidocaine, 0.5% levobupivacaine or 0.5% bupivacaine hydrochloride solutions. Viability was quantified using WST-1 and DNA assays. Viability and morphology were further characterised using CellTracker Green/Ethidium Homodimer-1 immunocytochemistry and function was assessed by an alkaline phosphatase assay. An additional group was cultured for a further seven days to allow potential recovery of the cells after removal of the local anaesthetic.

A statistically significant and dose dependent reduction in cell viability and number was observed in the cell cultures exposed to all three local anaesthetics at concentrations of 25% and 50%, and this was maintained even following culture for a further seven days.

This study indicates that certain local anaesthetic agents in widespread clinical use are deleterious to skeletal progenitor cells when studied in vitro; this might have relevance in clinical applications.

Currently, fibrin glue obtained from fibrinogen and thrombin of human and animal blood are widely investigated to use as injectable hydrogel for tissue engineering which contributes to minimally invasive surgery, superior biodegradability, cell attachment, proliferation and regenerating new tissue. However, most of them fail to achieve to be used for tissue engineering application because of a risk of immune response and poor mechanical properties. To overcome the limitation of fibrin glue and to reduce the usage of products from human and animal blood, the artificial fibrin glue materials were developed. Recently, cellulose nanofiber (CNF) as reinforcing agent has been explored for many tissue engineering applications such as bone and cartilage due to its impressive biological compatibility, biodegradability and mechanical properties. CNF was extracted from cassava pulp. PEO-PPO-PEO diacrylate block copolymer is a biodegradable synthetic polymers which is water insoluble hydrogel after curing by UV light at low intensity. To enhance the cell adhesion abilities, gelatin methacrylate (GelMA), the denature form of collagen was used to incorporate into hydrogel. The aim of this study was to develop the artificial fibrin glue from CNF reinforced PEO-PPO-PEO diacrylate block copolymer/GelMA injectable hydrogel. CNF/PEO-PPO-PEO diacrylate block copolymer/GelMA injectable hydrogels were prepared with 2-hydroxy-1-(4-(hydroxy ethoxy) phenyl)-2-methyl-1-propanone (Irgacure 2959) as a photoinitiator. The physicochemical properties were investigated by measuring various properties such as thickness, gel fraction, mechanical properties and water uptake. At optimal preparation condition, CNF reinforced injectable hydrogel was successful prepared after curing with UV light within 7 minutes. This hydrogel showed gel fraction and water uptake of 81 and 85%, respectively. The cytotoxicity, cell adhesion and proliferation of CNF reinforced injectable hydrogel was presented. Cellulose nanofiber from casava pulp was successfully used to prepare injectable hydrogel as artificial fibrin glue for tissue engineering. The hydrogel showed good physical properties which can be applied to use for tissue engineering application

A major obstacle in biofabrication is replicating the organization of the extracellular matrix and cellular patterns found in anisotropic tissues within bioengineered constructs. While magnetically-assisted 3D bioprinting techniques have the potential to create scaffolds that mimic natural biological structures, they currently lack the ability to accurately control the dispersion of magnetic substances within the bioinks without compromising the fidelity of the intended composite. To overcome this dichotomy, the concepts of magnetically- and matrix-assisted 3D bioprinting are combined here. This method preserves the resolution of printed structures by keeping low viscosity bioinks uncrosslinked during printing, which allows for the arrangement of magnetically-responsive microfibers without compromising the structural integrity of the design. Solidification is induced after the microfibers are arranged in the desired pattern. Furthermore, the precise design of these magnetic microfillers permits the utilization of low levels of inorganic materials and weak magnetic field strengths, which reduces the potential risks that may be associated with their use. The effectiveness of this approach is evaluated in the context of tendon tissue engineering, and the results demonstrate that combining the tendons like anisotropic fibrous microstructure with remote magneto-mechanical stimulation during in vitro maturation provides both biochemical and biophysical cues that effectively guide human adipose-derived stem cells towards a tenogenic phenotype In summary, the developed strategy allows the fabrication of anisotropic high-resolution magnetic composites with remote stimulation functionalities, opening new horizons for tissue engineering applications. Acknowledgments: ERC Grant CoG MagTendon nr 772817, BioChips PoC project nr 10106930, (PD/BD/129403/2017), (CEECIND/01375/2017), (2020.03410.CEECIND), (2022.05526.PTDC), (ED481B2019/025)

Tissue engineering and regenerative medicine are increasingly taking advantage of active materials, allowing to provide specific clues to the cells. In particular, the use of electroactive polymers that deliver electrical signals to the cells upon mechanical solicitation, open new scientific and technological opportunities, as they in fact mimic signals and effects present in living tissues, allowing the development of suitable microenvironments for tissue regeneration. In fact, electrical and electromechanical clues are among the most relevant ones in determining tissue functionality in tissues such as muscle and bone, among others, indicating their requirement for proper tissue regeneration. Piezoelectric polymers have already shown strong potential for novel tissue engineering strategies, once they can account for the existence of piezoelectricity within some specific tissues and also can modulate the electrical signals existing in tissue development and function. In this context, this talk reports on piezoelectric and magnetoelectric materials used for tissue engineering applications. The most used materials and morphologies for tissue engineering strategies are reported, together with the need of novel bioreactor designs allowing to take full advantage of those materials. Further, the main achievements, challenges and future needs for research and actual therapies will be presented and discussed

The key factors in Tissue Engineering are multipotent stem cells, growth factors (necessary to manipulate cell destiny) and scaffolds (3D constructs which support the growing tissue). Mesenchymal stem cells are the most important part of this equation, and it is procurement and manipulation of these that lies at the heart of tissue engineering. Luckily, mensenchymal stem cells can be obtained from many tissues, including synovium, bone marrow and periosteum. The use of bioreactors to optimise culture conditions and improve cell viability provides an opportunity to control stem cell destiny. Various Tissue Engineering strategies exist: manipulating cells in situ with osteogenic growth factors, such as BMP; implanting whole tissue grafts; and the use of Gene therapy. The tissues that concern orthopaedic surgeons are very diverse and no single tissue engineered construct will be able to fulfil all our clinical needs. Tissue engineering of articular cartilage is very difficult technically, but once accomplished will revolutionalise practice. The challenge lies in being able to produce cartilage as similar to native hyaline cartilage as possible. Although promising, ACI, using culture expanded cells, is able at best to produce hyaline-like cartilage but not the real thing. Multipotent mesenchymal stem cells are being used in this field. Even simply injecting these intraarticularly has been shown to retard the progression of OA in animal models. When attempting to regenerate meniscal cartilage, the mechanical properties of the scaffold become crucial, as the biomechanics of the knee are highly hostile. Ligaments and tendons, though the least complex tissues architecturally, have very high tensile properties which will be hard to replicate. The challenging aspects of Orthopaedic Tissue Engineering are manifold, yet the field itself is growing in leaps and bounds. Despite some initial setbacks, the new developments in this discipline are very encouraging

Tissue engineering in reconstructive surgery has many potential attractions, not the least to avoid donor site morbidity and reduce the potential need for allografts and prostheses. Currently there are only two products that have FDA approval in the United States, namely skin and cartilage. Other potential products being trialled are artificial blood vessels and heart valves. The common denominator of these is that they are essentially two dimensional and relatively avascular. Three dimensional tissue engineering has three essential components, (1) cells, (2) scaffold and (3) blood supply. Cells are most easily derived from an autologous source, by conventional tissue culture where they are expanded and implanted into the required site. They are committed cells and usually a large source of donor tissue is required to obtain an adequate source of cells for reconstruction. Stem cells have the potential to grow and differentiate, they may be embryonal which introduces ethical problems or adult stem cells. Cells can be genetically engineered to produce specific growth factors for the purpose of further cell proliferation, such as vascular endothelial growth factor for angiogenesis. The second essential is a scaffold for cells to adhere to and grow. This is particularly important for the development of the vascular network. Fibrin, PTFE (Dexon) Matrigel (a form of Laminen) or collagen are the most popular forms of matrix. The third and most essential component for three-dimensional tissue engineering is vascularization. To date, most tissue engineering research involves invitro studies of cell differentiation and growth but the invivo potential is limited because of inability to transfer a blood supply. At the Bernard O’Brien Institute at St Vincent’s Hospital, Melbourne, we have developed a model of invivo tissue engineering which involves the initial creation of a vascular core inside a plastic chamber which can be moulded to any desired shape. This construct seems to be an ideal environment for seeding of cells, including stem cells which allows them to survive and differentiate into various mesenchymal tissues. To date we have been able to generate skin flaps, fat, tissue and skeletal muscle. Although our prime interest has not been bone or cartilage it is reasonable to assume that this can be relatively simply produced in the same model from either stem cell sources or by the use of differentiating factors

Skeletal muscle tissue engineering has made progress towards production of functional tissues in line with the development in materials science and fabrication techniques. In particular, combining the specificity of 3D printing with smart materials has introduced a new concept called the 4D printing. Inspired by the unique properties of smart/responsive materials, we designed a bioink made of gelatin, a polymer with well-known cell compatibility, to be 3D printed on a magnetically responsive substrate. Gelatin was made photocrosslinkable by the methacrylate reaction (GELMA), and its viscosity was finetuned by blending with alginate which was later removed by alginate lyase treatment, so that the printability of the bioink as well as the cell viability can be finetuned. C2C12 mouse myoblasts-laden bioink was then 3D printed on a magnetic substrate for 4D shape-shifting. The magnetic substrate was produced using silicon rubber (EcoFlex) and carbonyl iron powders. After 3D printing, the bioink was crosslinked on the substrate, and the substrate was rolled with the help of a permanent magnet. Unrolled (Open) samples were used as the control group. The stiffness of the bioink matrix was found to be in the range of 13–45 kPa, which is the appropriate value for the adhesion of C2C12 cells. In the cell viability analysis, it was observed that the cells survived and could proliferate within the 7-day duration of the experiment. As a result of the immunofluorescence test, compared to the Open Group, more cell nuclei were observed overlapping MyoD1 expression in the Rolled Group; this indicated that the cells in these samples had more cell-cell interactions and therefore tended to form more myotubes. Acknowledgements: This research was supported by the TÜBİTAK 2211-A and YÖK 100/2000 scholarship programs

Successful anterior cruciate ligament (ACL) reconstructions strive a firm ligament-bone integration. Therefore, the aim of this study was to address in more detail the enthesis as the thriphasic bone attachment of the ACL using a tissue engineering approach. To establish a tissue-engineered enthesis-like construct, triphasic scaffolds embroidered from poly(L-lactide-co-caprolactone) and polylactic acid functionalized with collagen foam were colonized with osteogenically differentiated human mesenchymal stromal cells (hMSCs) and lapine (L) ACL fibroblasts. These triphasic scaffolds with a bone-, a fibrocartilage transition- and a ligament phase were seeded directly after spheroid assembly or with 14 days precultured LACL fibroblast spheroids and 14 days osteogenically differentiated hMSCs spheroids (=longer preculture) and cultured for further 14 days. Cell survival was tested. Collagen type I and vimentin were immunolabeled and the content of DNA and sulfated glycosaminoglycan (sGAG) was quantified. The relative gene expression of tenascin C, type I and X collagens, Mohawk and Runx2 was analyzed. Compared to the LACL spheroids the hMSC spheroids adhered better to the scaffold surface with faster cell outgrowth on the fibers. Collagen type I and vimentin were mainly detected in the hMSCs colonizing the bone zone. The DNA content was generally higher in the bone (hMSCs) than in the ligament zones and after short spheroid preculture higher than after longer preculture whereas the sGAG content was greater after longer preculture for both cell types. The longer precultivated hMSCs expressed more type I collagen in comparison to those only shortly precultured before scaffold seeding. Type I collagen and tenascin C were higher expressed in scaffolds directly colonized with LACL compared to those seeded after longer spheroid preculture. The gene expression of ECM components and transcription factors depended on cell type and preculturing condition. Zonal colonization of triphasic scaffolds using the spheroid method is possible offering a novel approach for enthesis tissue engineering

Background. Auxetic materials have a negative poisons ratio, and a number of native biological tissues are proposed to possess auxetic properties. One such tissue is annulus fibrosus (AF), the fibrous outer layers of the intervertebral disc (IVD). However, few studies to date have investigated the potential of these materials as tissue engineering scaffolds. Here we describe the potential of manually converted polyurethane (PU) foams as three dimensional cellular scaffolds for AF repair. Methods. Rat MSCs were seeded onto fibronectin coated auxetic foams at a cell density of 6.4 × 10. 3. cells/mm. 3. , and cultured for up to 3 weeks. Cell viability was assessed throughout culture and following culture scanning electron microscopy (SEM) was used to assess morphological characteristics. Histological assessment was performed to assess production of matrix proteins. Results. Cells adhered to the surface auxetic foams and remained viable for the 3 weeks investigated. Histology and SEM demonstrated cells within the full thickness of the auxetic foams, where extracellular matrix was starting to be produced following 3 weeks, including collagens suggesting differentiation of the MSCs. Conclusion. Auxetic PU foams have a significant potential for use in tissue engineering applications, potentially mimicking the multiaxial strains of annulus fibrous tissue. MSCs were shown to adhere, survive and produce matrix within the foams after 3 weeks, future work will focus on longer term studies and in depth analysis of the phenotype of the cells. No conflicts of interest. Funding provided by a grant from Sheffield Children's Hospital NHS trust

Purpose: The purpose of our tissue engineering work was to produce a substitute for the anterior cruciate ligament (ACL) in laboratory cultures for human implantation and to conduct fundamental studies on healing mechanisms. Material: We used cells isolated from ACL biopsies obtained from the host, type I bovine collagen, and two bone blocks to produce ACL in culture. Methods: Several layers of collagen containing host autologous ACL cells were superposed and linked to two bones that were placed on either side, according to a process currently being patented. The cells, or fibroblasts, enter into contact with the collagen matrix and start remodelling it, in the laboratory, before implantation. This ACL produced by tissue engineering can be ready for implantation 10–12 days after isolating the autologous cells from a ruptured ACL. Results: Implantation of autologous ACL reconstructs was successful in eight goats. Histological analysis of the implanted grafts showed permanent integration into the tissues after 1–13 months. Th synovial membrane was reformed and rapidly vascularised, about one month after the graft. Thereafter, remodelling of the collagen matrix led to the formation of a very dense network of fibres, organised in bundles, very comparable to the normal histological aspect of the ACL. The bone blocks were also integrated by incorporation into the femur and tibia of the host. Sharpey fibres were present at the bone-ligament surface and a well structured fibro-cartilage was observed. In addition, the synovial membrane around the graft was innervated five months after implantation, suggesting that propioception could be recovered over time. Finally, progressive gain in force reached 20 – 36% of the normal ACL, 9 to 13 months after implantation;. Discussion: These promising data demonstrate that an autologous ACL with an interesting potential for regeneration can be produced in the laboratory, avoiding the risk of rejection and sparing healthy knee structures, thus favouring more rapid functional rehabilitation. Conclusion: Tissue engineering is a new avenue of research with potential applications in orthopaedic surgery, particularly for reconstruction of the ACL

Organ and tissue decellularisation are promising approaches for the generation of scaffolds for tissue regeneration since these materials provides the accurate composition and architecture for the specific tissues. Repopulation of the devitalized matrixes is the most critical step and a challenge, especially in dense tissues such as cartilage. To overcome this difficulty, several chemical and mechanical strategies have been developed. Chemical extraction targeting specific matrix components such as elastin, makes auricular cartilage accessible for cells via channels originating from the elastic fiber network. However, chemical treatment for glycosaminoglycan removal is not sufficient to allow cell ingrowth in articular cartilage. As alternative, laser perforation has been developed allowing to engrave fine structures with controlled size, distance and depth, with reproducibility and high throughput. Two of the most commonly used laser technologies used in the medical field, the CO. 2. and femtosecond laser, were applied to hyaline cartilage with very different structural effect. Within this talk, the structuralizing possibilities of laser and enzymatic treatments, the effect on the matrix and the general advantages and disadvantages for tissue engineering are discussed. We believe that the optimal combination of chemical and laser treatment has high potential for a new generation of biomaterials for tissue engineering

Bottom-up tissue engineering (TE) strategies employing microscale living materials as building blocks provide a promising avenue for generating intricate 3D constructs resembling native tissues. These microtissue units exhibit high cell densities and a diverse extracellular matrix (ECM) composition, enhancing their biological relevance. By thoughtfully integrating different cell types, the establishment of vital cell-cell and cell-matrix interactions can be promoted, enabling the recreation of biomimetic micro-niches and the replication of complex morphogenetic processes. Notably, by co-assembling blood vessel-forming endothelial cells with supportive stromal cells, microtissues with stable capillary beds, referred to as vascular units (VUs), can be generated. Through a modular TE approach, these VUs can be further combined with other microtissues and biomaterials to construct large-scale vascularized tissues from the bottom up. Integration of VUs with technologies such as 3D bioprinting and microfluidics allows for the creation of structurally intricate and perfusable constructs. In this presentation, we will showcase examples of VUs and explore their applications in regenerative medicine and tissue modeling. Acknowledgements: This work was supported by project EndoSWITCH (PTDC/BTM-ORG/5154/2020) funded by FCT (Portuguese Foundation for Science and Technology)

By definition, a smart biomaterial is a material, such as a ceramic, alloy, gel or polymer, that can convert energy from one form into another by responding to a change in a stimulus in its environment. These stimuli may involve temperature, pH, moisture, or electric and magnetic fields. In particular, thermoresponsive biomaterials have been successfully employed to host mammalian cells with a view to musculoskeletal tissue engineering. The presentation provides an overview of the use of thermosensitive polymers for the non-enzymatic stem cell harvesting, cell sheet engineering, three-dimensional scaffolds fabrications and organ-printing materials

Tissue engineering is a promising approach to regenerate damaged skeletal tissues. In particular, the use of injectable hydrogels alleviates common issues of poor cell viability and engraftment. However, uncontrolled cell fate, resulting from unphysiological environments and degradation rates, still remain a hurdle and impedes tissue healing. We thus aim at developing a new platform of injectable hyaluronic acid (HA) hydrogels with a large panel of properties (stiffness, degradation…) matching those of skeletal tissues. Hence, HA with different molecular weights were functionalized with silylated moieties. Upon injection, these hydrogels formed through a sol-gel chemistry within 5 to 20 minutes in physiological conditions, as demonstrated by rheological characterization. By varying the crosslinking density and concentration, we obtained hydrogels spanning a large range of elastic moduli (E = 0.1–20 kPa), similar to those of native ECMs, with tunable biodegradation rates (from 24 hours to > 50 days) and swelling ratios (500 to 5000% (w/w)). Cell viability was confirmed by Live/Dead assays and will be completed by in vivo subcutaneous implantations in mice to study the foreign body reaction and degradation rate. We further developed hybrid HA/biphasic calcium phosphate granules hydrogels and demonstrated a strong mechanical reinforcement (E = 0.1 MPa) and a faster relaxation behaviour (τ. 1/2. < 400s), with similar degradation rates. Ongoing in vitro differentiation assays and in vivo implantations in a rabbit femur model will further assess their ability to drive bone regeneration. Collectively, these results suggest that this hydrogel platform offers promising outcomes for improved strategies in skeletal tissue engineering

Current issues being debated in ACL reconstruction include injury prevention, graft choice, graft positioning, graft fixation, graft remodelling and rehabilitation. Tissue engineering, the alteration of biological mechanisms by application of novel proteins, enzymes and hormones, is rapidly changing the way we approach all aspects of surgery. Tissue engineering techniques in ACL/PCL reconstruction focus on new biosynthetic ACL material, fixation of soft tissue grafts to bony tunnels and graft remodelling. OP-1 is recombinant human Osteogenic Protein 1 (BMP-7). It is a member of the Transforming Growth Factor β (TGFβ) super family. OP-1 promotes the recruitment, attachment, proliferation and differentiation of pluripotential mesenchymal stem cells. It promotes both osteogenesis and chondrogenesis. The carrier is highly purified bovine bone type 1 collagen, which provides an osteoconductive matrix. We have completed a study assessing the use of OP-1 as a means of enhancing early biological fixation of soft tissue grafts within bone tunnels in a sheep ACL model. We have commenced a clinical trial using OP-1 in adult ACL reconstruction, believing that OP-1 will enhance early biological graft fixation, and hence, improve clinical results, speed up rehabilitation and prevent tunnel widening. Other studies have shown the beneficial effects of BMP-2 on an extraarticular bone tendon fixation model, the use of TGF-B to enhance graft remodelling and the application of gene therapy to deliver BMP’s for enhanced graft fixation. Several projects are underway looking at creating biosynthetic ACL grafts using tissue engineering techniques. As opposed to purely synthhetic grafts, bioACL grafts are made of a collagen scaffold, allowing for remodelling and revascularisation. ACL reconstructive surgery is constantly evolving. Tissue engineering may provide us with a means of minimising morbidity, accelerating rehabilitation and improving the clinical outcome following this common surgery

Biodegradable porous scaffolds play an important role in tissue engineering as the temporary templates for transplanted cells to guide the formation of the new organs. The most commonly used porous scaffolds are constructed from two classes of biomaterials. One class consists of synthetic biodegradable polymers such as poly (α-hydroxy acids), poly(glycolic acid), poly(lactic acid), and their copolymer of poly(DL-lactic-co-glycolic acid) (PLGA). The other class consists of naturally derived polymers such as collagen. These biomaterials have their respective advantages and drawbacks. Therefore, hybridization of these biomaterials has been expected to combine their advantages to provide excellent three-dimensional porous biomaterials for tissue engineering. Our group developed one such kind of hybrid biodegradable porous scaffolds by hybridizing synthetic poly (α-hydroxy acids) with collagen. Collagen microsponges were nested in the pores of poly (α-hydroxy acids) sponge to construct the poly (α-hydroxy acids)-collagen hybrid sponge. Observation by scanning electron microscopy (SEM) showed that microsponges of collagen with interconnected pore structures were formed in the pores of poly (α-hydroxy acids) sponge. The mechanical strength of the hybrid sponge was higher than those of either poly (α-hydroxy acids) or collagen sponges both in dry and wet states. The wettability with water was improved by hybridization with collagen, which facilitated cell seeding in the hybrid sponge. Use of the poly (α-hydroxy acids) sponge as a skeleton facilitated formation of the hybrid sponge into the desired shapes with high mechanical strength, while collagen microsponges contributed good cell interaction and hydrophilicity. One of such kind of hybrids. Additionally, our group developed a hydrostatic pressure bioreactor for chondrocyte culture. And our study showed that hydrostatic pressure (0–3 MPa) had promotional effects on the production of proteoglycan and type II collagen by cultured chondrocytes. Therefore, it would be a promising pathway for reconstructing cartilage-like tissue to culture chondrocytes in this three-dimensional hybrid sponge under physiological hydrostatic pressure

Tissue engineering regards the generation, regeneration, augmentation or limitation of the structure and function of living tissues by the application of scientific and engineering principles. Skeletal defects resulting from tumor resection, congenital abnormalities or trauma often require surgical intervention to restore the function. Current option for bone replacement include autografts,allografts,metals,ceramic and polymers.However, all these materials have drawbacks, and their selection usually require some compromises. Skeletal tissues are under extensive investigation in tissue engineering research and beside the biological issues, the scaffolds design plays an important role. A number of biodegradable and bioabsorbable materials as well as scaffold designs, have been experimentally and, in some cases clinically studied. An appropriate scaffold should posses highly porous with interconnected pore network for cell growth and flow transport of nutrient and metabolic waste; biocompatible and bioresorbable with a controlled degradation and resorption rate to match cell/ tissue growth, suitable surface chemistry for cell attachment, proliferation and differentiation, and mechanical properties to match those of the implanted tissue. Synthetic biodegradable polymers and inorganic materials are promising as extracellular matrix analogue to facilitated tissue development and growth; these include: polyglycolic acid, poly-l-lactic acid, copolymers, poly-caprolactones, hydroxyapatite, tricalcium phosphates. All these scaffolds are well performing from biological and chemical-physical but they have some limitations from mechanical point of view. To overcome this problem a composite structure made by Polycaprolactone and Hydroxyapatite is studied by mechanical and biological analysis. To obtain a porous structure, the casting and salt leaching technique is implemented. The composite shows mechanical properties in the range of the spongy bone and interesting biological properties with regards to osteoblasts. Injectable gels made of collagen are analysed to carry cells, a preliminary results of collagen gel loaded with MSC cells have been performed and rheological and proliferation study are showing the feasibility to obtain a bioactive materials/cells to be inject in the defined body site defects avoiding massive surgery

Hyaline cartilage and immature nucleus pulposus possess similar macromolecules in their extracellular matrix, and there is no unique molecular marker to distinguish the two tissues. We show that in normal disc (fifteen to twenty-five years old), the GAG to hydroxyproline ratio (proteoglycan to collagen ratio) within the nucleus pulposus is approximately 28:1. However, the GAG to hydroxyproline ratio within hyaline cartilage of the same group is 2.5:1. This information is important in identifying stem cell conversion to a nucleus pulposus cell phenotype rather than a chondrocyte phenotype for tissue engineering of intervertebral disc. Tissue engineering of intervertebral discs (IVDs) using mesenchymal stem cells (MSCs) induced to differentiate into a disc-cell phenotype has been considered as an alternative treatment for disc degeneration. Since there is no unique marker for disc tissue, and because cartilage and immature nucleus pulposus (NP) possess similar macromolecules in their extracellular matrix, it is currently difficult to recognize MSC conversion to a disc cell. In this study, we compare the proteoglycan to collagen ratio in the NP of normal disc to that of the hyaline cartilage of the endplate within the same group of individuals. To distinguish between a normal NP and hyaline cartilage phenotype for tissue engineering of IVDs. Human lumbar spine specimens were harvested from fresh cadavers, aged twelve week to seventy-nine year. Discs and endplates were examined for total collagen using the hydroxyproline assay and glycosaminoglycan (GAG) content using a standard assay. In a mature disc with no degeneration (fifteen to twentyfive years), the GAG to hydroxyproline ratio within the NP is approximately 28:1. However, the ratio within the hyaline cartilage endplate of the same group is 2.5:1. A high proteoglycan to collagen ratio can be used to distinguish NP cells from chondrocytes. The lower NP collagen content is probably responsible for its gelatinous nature rather than the firm texture of hyaline cartilage, and this is essential for normal disc function. This information is crucial in identifying a NP-like phenotype when MSCs are induced to differentiate into a disc cell as opposed to a chondrocyte, for tissue engineering of IVDs

Collagen materials are extensively used in regenerative medicine. However, they still present limitations such as a mono-domain composition and poor mechanical properties. On the other hand, tissue grafts overcome most of these limitations. In addition, the potential of tissue grafts in musculoskeletal tissue engineering has not been fully investigated. Herein, we ventured to assess the potential of a decellularised porcine peritoneum for musculoskeletal applications by comparing its characteristics with a commercial collagen scaffold employed in tendon. Results indicated that the porcine peritoneum had higher mechanical properties and a lower crosslinking ratio (p < 0.01). Furthermore, it presented a lower resistance to collagenase digestion, which suggests a faster remodelling in vivo of the tissue graft. Immunohistochemistry analysis showed a preserved and multicomponent structure in the porcine peritoneum contrary to the collagen matrix, confirming the multifunctional nature of the tissue graft. Regarding the cell-response assessment, tenocytes and ADSCs were able to grow on both materials, however, proliferation was enhanced by the porcine peritoneum (p<0.01). Immune response by THP-1 showed an acute inflammatory response by macrophages to the collagen matrix, contrary to that observed in the porcine peritoneum which triggered a mild reaction. The in-progress in vivo study in a rabbit tendon model will elucidate the potential of porcine peritoneum for tendon repair applications. The present study shows how the multifunctionality of the porcine peritoneum provides higher cytocompatibility than a mono-domain collagen matrix with human tenocytes and ADSC. Besides, its lower immune response in vitro suggests better remodelling after implantation

Osteoarthritis (OA) is one of the most prevalent joint diseases involving progressive and degenerative changes to cartilage resulting from a variety of etiologies including post-traumatic incident or aging. OA lesions can be treated at its early stages through cell-based tissue engineering therapies using Mesenchymal Stem Cells (MSCs). In vivo models for evaluating these strategies, have described both chondral (impaction) and osteochondral (biopsy punch) defects. The aim of the investigation was to develop a compact and reproducible defect inducing post-traumatic degenerative changes mimicking early OA. Additionally, a pilot study to evaluate the efficacy of MSC-hydrogel treatment was also assessed. Surgery was performed on New Zealand white rabbits (male, 5–8 months old) with defects created on medial femoral condyle. For developing an appropriate defect, three approaches were used for evaluation: a biopsy punch (n = three at six and twelve weeks), an impaction device1 (n = three at six and twelve weeks) and a dental drill model (n = six at six and twelve weeks). At stated time points, condyles were harvested and decalcified in 10% EDTA, then embedded in Tissue-Tek and sectioned using a cryostat. Upon identification of region of interest, sections were stained with Safranin-O/Fast green and scored using OARSI scoring system by two blinded observers2. For the pilot study, autologous bone marrow was harvested from rabbits and used to isolate and expand MSCs. The Dental drill model was applied to both knee condyles, left untreated for six weeks at which stage, PKH26 fluorescently labelled MSCs were seeded into a hyaluronic acid hydrogel (TETEC). Repair tissue was removed from both condyles and MSC-hydrogel was injected into the left knee, whilst right knee was left empty. Rabbits were sacrificed at one (n = 1), six (n = 3) and twelve (n = 3) weeks post-treatment, processed as previously described and cartilage regeneration evaluated using Sellers score3. Impacted condyles exhibited no observed changes histologically (Mean OARSI score = 1 + 1), whereas biopsy punched and dental drilled defects demonstrated equal signs of cartilage erosion (OARSI score = 3 + 1) at assessed time points. However, biopsy punched condyles formed a diffusive defect, whereas dental drilled condyles showed a more defined, compact and reproducible defect. In the pilot study, PKH-labelled MSCs were observed at one and six weeks post-implantation within the defect space where hydrogel was injected. Tissue regeneration assessment indicated no difference between empty (Mean Sellers score = 14 + 2) and MSC treated defects (Sellers score = 16 + 5) at six weeks post-injection. At twelve weeks, MSC treated defects showed improved tissue regeneration with substantial subchondral bone restoration and good integration of regenerative cartilage with surrounding intact tissue (Sellers score = 10 + 1), whereas untreated defects showed no change in regeneration compared to six weeks (Sellers score = 16 + 2). Dental drill model was found to be the appropriate strategy for investigating early OA progression and treatment. Application of MSCs in defects showed good cartilage regeneration after twelve weeks application, indicating their promise in the treatment of early OA defects

Long-term regeneration of cartilage defects treated with tissue engineering constructs often fails because of insufficient integration with the host tissue. We hypothesize that construct integration will be improved when implants actively interact with and integrate into the subchondral bone. Growth and Differentiation Factor 5 (GDF-5) is known to support maturation of chondrocytes and to enhance chondrogenic differentiation and hypertrophy of mesenchymal stromal cells (MSC). Therefore, we investigated whether GDF-5 is capable to stimulate endochondral ossification of MSC in vitro and in vivo and would, thus, be a promising candidate for augmenting fibrin glue in order to support integration of tissue engineering constructs into the subchondral bone plate. To evaluate the adhesive strength of fibrin glue versus BioGlue. ®. , a commercially available glue used in vascular surgery, an ex vivo cadaver study was performed and adhesion strength was measured via pull-out testing. MSC were suspended in fibrin glue and cultivated in chondrogenic medium with and without 150 ng/mL GDF-5. After 4 weeks, the formed cartilage was evaluated and half of the constructs were implanted subcutaneously into immunodeficient mice. Endochondral ossification was evaluated after 2 and 4 weeks histologically and by microCT analysis. BioGlue. ®. and GDF-5-augmented fibrin glue were tested for 4 weeks in a minipig cartilage defect model to assess their orthotopic biocompatibility. Pull-out testing revealed sufficient adhesive strength of fibrin glue to fix polymeric CellCoTec constructs in 6 mm cartilage defects, however, BioGlue. ®. showed significantly higher adhesive power. In vitro chondrogenesis of MSC under GDF-5 treatment resulted in equal GAG deposition and COLIIa1 and ACAN gene expression compared to controls. Importantly, significantly increased ALP-activity under treatment with GDF-5 on day 28 indicated enhanced hypertrophic differentiation compared to controls. In vivo, MSC-fibrin constructs pre-cultured with GDF-5 developed a significantly higher bone volume on day 14 and 28 compared to controls. When pre-cultured with GDF-5 constructs showed furthermore a significantly higher bone compactness (bone surface/bone volume coefficient) than controls, and thus revealed a higher maturity of the formed bone at 2 weeks and 4 weeks. Orthotopic biocompatibility testing in minipigs showed good defect filling and no adverse reactions of the subchondral bone plate for defects treated with GDF-5-augmented fibrin glue. Defects treated with BioGlue. ®. , however, showed considerable subchondral bone lysis. Thus, BioGlue. ®. – despite its adhesive strength – should not be used for construct fixation in cartilage defects. GDF-5-augmented fibrin glue is considered promising, because of a combination of the adhesive strength of fibrin with an enhanced osteochondral activity of GDF-5 on MSC. Next step is to perform a large animal study to unravel whether GDF-5 stimulated endochondral ossification can improve scaffold integration in an orthotopic cartilage defect model

Introduction: The anterior cruciate ligament (ACL) is the most frequently damaged ligament in the knee joint. The patella tendon autograft is the current replacement of choice, however autografts are not always available and grafting often leads to donor site morbidity. Allogeneic implants may cause an adverse immunological reaction [. 1. ] The aim of this study was to develop an acellular tendon scaffold with the mechanical and biochemical properties of tissue which could be rapidly recellularised for use in tissue engineering of the anterior cruciate ligament. Materials and Methods: Porcine patella tendons were dissected less than 24 hours after slaughter and washed in PBS. The tendons were decellularised using 0.1% (w/ v) SDS for 24 hours. Decellularisation was assessed by haematoxylin and eosin staining and light microscopy. The glycosaminoglycan and hydroxyproline (measure of collagen) content of the scaffold were also assessed quantitatively following decellularisation. Following decellularisation the scaffolds were subject to various levels of ultrasonication in order to modify the acellular scaffold prior to reseeding in an attempt to achieve recellularisation of the scaffold. Denaturation of the collagen within the scaffold following ultrasonication was assessed using the ƒÑ-chymotrypsin assay. Decellularised and ultrasonicated scaffolds were subject to uniaxial tensile loading to failure in a Howden tensile testing machine. The sonicated scaffolds were reseeded with human tenocytes (1x105 cells.cm2) and cultured in 5% CO2 in air at 37°C for three weeks. One scaffold was removed every seven days and either fixed in 10% neutral buffered formalin prior to dehydration and H& E staining or was stained with Live/Dead stain (Molecular Probes) and observed using confocal microscopy. Results: Porcine patella tendons were successfully decellularised using 0.1% (w/v) SDS. Following decellularisation there was no change in the biochemical composition of the scaffold. Ultrasonication of the scaffold at 360W was shown to open up spaces between collagen bundles without damaging the collagen matrix and this was confirmed with the ƒÑ-chymotrypsin assay. Following decellularisation and ultrasonication there was no change in the ultimate force (N) needed to break the tendon scaffold. When cells were seeded onto the sonicated scaffold, the cells were shown to penetrate to the centre of the scaffold within just 3 weeks of culture. Following staining with Live/Dead stain it was shown that after three weeks in static culture approximately 50% of the cells in the centre of the scaffold were viable. In comparison the cells cultured on the acellular non-sonicated scaffold remained on the surface of the scaffold and did not penetrate the matrix during this culture period. Conclusion: An acellular scaffold with excellent biochemical and mechanical properties has been developed which can be recellularised in an important first step towards tissue engineering of the anterior cruciate ligament. Future work will investigate culture of the reseeded scaffold under appropriate physical stimulation with a view to maintaining tissue homeostasis and increasing cell viability

Aims: The aim of this research work was the realization of an inorganic bioactive scaffold for bone regeneration. This biomaterial should be macroporous, in order to allow the bone in-growth, and bioactive aiming to promote the bone regeneration and healing. Methods: The macroporous biomaterial was prepared by consolidation of a suspension of starch and SiO2-CaO-Na2O-MgO glass powders. Starch powders were used as both pore former and consolidation agent. Starch-glass green bodies were prepared by uniaxial pressing and, after drying, they were heated to remove the organic phase and to sinter the inorganic one. The sintered scaffolds were characterized by X-Ray diffraction, scanning electron microscopy and mercury intrusion porosimetry. The scaffolds bioactivity was evaluated soaking the samples in a simulated body fluid for periods up to 4 weeks. On the most representative samples, in vitro tests of adhesion and proliferation were performed using human primary osteoblast-like cells. Results: The obtained scaffolds showed an interconnected macroporosity of 50–100 B5m and a satisfactory degree of sintering. The sintering treatment induced the nucleation and growth of Na2Ca2(SiO2)3 crystals which is a phase that possess a very high bioactivity index. By soaking the scaffolds in SBF for period up to 1 month, an extensive precipitation of hydroxylapatite, with the typical globular morphology, occurred both inside and outside the pores. The adhesion and proliferation tests showed a remarkable spreading of the osteoblasts on the scaffold surface and thus a good biological response. Conclusions: Scaffolds with interconnected porosity were successfully obtained. The pores are highly interconnected and homogenously distributed in the samples. The chosen thermal treatment and the use of starch powders led to a final macroporous glass-ceramic structure. The obtained scaffolds showed a very high in vitro bioactivity with precipitation of HAp. Moreover, preliminary biological tests, showed a satisfactory cellular interaction with the proposed biomaterials. For the above-mentioned reasons, the starch consolidation method, the optimized processing parameters and the tailored glass composition can be used to produce scaffolds suitable for bone substitutions and tissue engineering

During the last decade there has been an increasing interest in the management of cartilage lesions, owing to the introduction of new therapeutic options. Beside the improvement of the classical vascular techniques (mosaicplasty, microfractures, etc.), cell therapy and tissue engineering have opened new perspectives in this field. One of the most recent tissue engineering techniques is represented by the MACI‚ (Matrix-induced Autologous Chondrocyte Implantation). This method requires seeding of autologous chondrocytes on a type I-III collagene membrane, after their arthroscopy harvesting from the knee and subsequent in vitro expansion of the cellular population using autologous serum. The seeded membrane is implanted in the chondral defect using exclusively fibrin glue, through a limited exposure joint approach. Membrane structure and its cellular population were investigated by light microscopy, SEM and electrophoresis (SDS PAGE 7%) before implantation. There was evidence of chondroblasts and type II collagen inside the seeded membrane. Clinical series. At the Institute of Orthopaedics and Traumatology of the University of Insubria in Varese (Italy), the MACI‚ technique was used for the treatment of 13 patients, affected by chondral defects, between December 1999 and January 2001. There were 9 males and 4 females with an average age of 35 years (range, 18 to 49 years). The sites of the defects were the following: 8 medial femoral condyle, 2 lateral femoral condyle, 1 femoral trochlea, 2 talar dome. The average size of the defects was 3.5 cm2 (range, 2 to 4.5 cm2). The clinical and functional evaluation was performed using the ICRS (International Cartilage Repair Society) rating scale, the modified Cincinnati rating system, Lysholm II and Tegner scores for the knee, while the AOFAS (American Orthopaedic Foot and Ankle Society) score was used for the ankle. MRIs were taken before the operation as well as at 6 and 12 months postoperatively. The average follow-up was 6.5 months (range, 2 to 15 months). No complications were observed in the postoperative period. The six patients with a minimum follow-up of 6 months showed an improvement in the clinical and functional status after the operation, as testified by the scores reached with the different rating systems used. MRIs showed the presence of hyaline-like cartilage at the site of implantation. Conclusions. According to our preliminary experience, the MACI‚ technique offers several advantages (technical simplicity, short operating times, minimal invasivity and easier access to difficult sites) and appears a reliable method for the repair of deep cartilage defects

The selection of a proper material to be used as a scaffold or as a hydrogel to support, hold or encapsulate cells is both a critical and a difficult choice that will determine the success of failure of any tissue engineering and regenerative medicine (TERM) strategy. We believe that the use of natural origin polymers, including a wide range of marine origin materials, is the best option for many different approaches that allow for the regeneration of different tissues. In addition to the selection of appropriate material systems it is of outmost importance the development of processing methodologies that allow for the production of adequate scaffolds/matrices, in many cases incorporating bioactive/differentiation agents in their structures. An adequate cell source should be selected. In many cases efficient cell isolation, expansion and differentiation, and in many cases the selection of a specific sub-population, methodologies should be developed and optimized. We have been using different human cell sources namely: mesenchymal stem cells from bone marrow, mesenchymal stem cells from human adipose tissue, human cells from amniotic fluids and membranes and cells obtained from human umbilical cords. The development of dynamic ways to culture the cells and of distinct ways to stimulate their differentiation in 3D environments, as well as the use of nano-based systems to induce their differentiation and internalization into cells, is also a key part of some of the strategies that are being developed in our research group. The potential of each combination materials/cells, to be used to develop novel useful regeneration therapies will be discussed. The use of different cells and their interactions with different natural origin degradable scaffolds and smart hydrogels will be described. Several examples of TERM strategies to regenerate different types of musculoskeletal tissues will be presented. Relevance to orthopaedics will be highlighted

The use of stem cells in tissue engineering has emerged as a promising therapy for the repair of bone and cartilage defects. Targeted delivery of stem cells requires a substrate to maintain the cells at the repair site, as well as to provide the physical cues, such as mechanical strain, for encouraging differentiation and expression of the mature cell phenotype. The strains that will be generated in cells residing on the scaffold is dependent on the scaffold material, as well as both the fibre thickness and the fibre orientation in the scaffold. To encourage uniform bone matrix generation throughout the scaffold, it is desirable that the strain be uniformly distributed and that the internal pore architecture be precisely controlled to maximise media diffusion. This requires an optimised scaffold design and a manufacturing technique that allows for precise control over the scaffold’s internal architecture. Scaffold architecture was optimised by performing a series of finite element analyses (FEA) on computer aided design (CAD) models of Polycaprolactone (PCL) scaffolds. The mechanical properties of PCL were used to yield an accurate strain profile of scaffolds with different fibre orientations. Having determined the optimal scaffold geometry, PCL scaffolds were manufactured using a fibre deposition technique that yielded three-dimensional objects with this geometry. During manufacture, a PCL solution was extruded into a non-miscible solvent which precipitated out PCL fibres in repetitive layers. Of the geometries tested with FEA, a 90 degree rotation of adjacent layers with a 50% offset of parallel strands was found to provide the optimal strain distribution (60% increase in surface exposed to strain). Histomorphometry was used to assess the exact dimensions of the scaffold produced. Fibre spacing was found to be precisely controlled to 380 +/- 10 microns within the layers and the fibre thickness was controlled to 270 +/- 10 microns. This demonstrates that FEA can be used to predict the strain distribution of different CAD models and that the fibre deposition solvent extrusion technique can be used to accurately manufacture PCL scaffolds that match the desired architecture

Tissue engineering is founded on the principle of pro-actively manipulating the triad of tissue regeneration. The triad consists of matrices, pluripotential cells and signaling factors. Our hypothesis is that advances in orthopedic surgery to successfully regenerate bone are accomplished by incorporating optimised matrices into the surgeon’s armamentarium. Pro Osteon is a bioactive ceramic matrix with interconnected porosity. It has been evaluated in experimental animals and used clinically as a bone graft substitute for more than two decades. It is available in slowly resorbable form composed of hydroxyapatite and as a more rapidly resorbable composite of calcium carbonate and calcium phosphate. Experiments have been conducted in sheep, rats and dogs to demonstrate consistent and predictable bone regeneration when the implant is placed in direct apposition to host bone, the host bone is viable and the interfaces between the bone and implants are biomechanically stable. Most importantly, controlled, multi-center clinical trials showed consistent efficacy and safety in humans. Either as a block or granules, Pro Osteon is biocompatible and osteophilic and osteoconductive. Bone regeneration, as demonstrated radiographically and histologically, occurs directly within the porous ceramic in traumatic defects and tumors. Where surrounding viable bone or mechanical stability is inadequate, such as posterior spinal fusion, the ceramic must be co-mixed with autograft. For indications where autograft is limited or unavailable, bone regeneration within the porosity was enhanced and fusion achieved by supplementing Pro Osteon with bone marrow and/or with growth factors. This was demonstrated experimentally and clinically. Mitogenic and/or morphogenic growth factors were demonstrated to increase the rate or degree of bone formation. Methods and equipment for intra-operative collection of concentrated platelets were shown to be a cost-effective and safe source of autologous mitogens. Using a variety of ectopic and orthotopic animals models, we have shown that autologous, purified xenogenic and recombinant growth factors will bind to the surface of Pro Osteon and initiate or stimulate the bone induction process. In conclusion, Pro Osteon is an effective matrix for bone formation. It can be used alone or it can be used in combination with pluripotential, osteogenic stem cells or with signaling proteins

Introduction: The aim of this study is to develop a novel approach to tissue engineering in vivo, in which the adaptive response of skeletal tissues to the imposed mechanical environment will be utilised to induce a cartilaginous resurfacing of the acetabular articulation in a hemi-arthroplasty model of hip replacement. Our hypothesis was that a cartilaginous resurfacing of subchondral bone can be induced by applying stresses of 0 to 3 MPa to the articular surface of the acetabulum. We used an ovine hemiarthroplasty model where the stresses on the acetabulum were engineered by using different femoral head sizes. Methods: Three groups of six sheep received unilateral hip hemi-arthroplasties and were sacrificed 24 weeks post-operatively to harvest the acetabula. At operation, acetabular cartilage was removed completely and the subchondral bone was reamed down and left bleeding. Three femoral head sizes, 25, 28, and 32-mm, were used to induce different contact stress levels. Vertical ground reaction force (GRF) data were measured and normalised by body weight for both limbs pre-operatively and every 4 weeks post-operatively. Five specimens from each group and eight unoperated controls were processed and stained with Safranin O and Sirius Red. Cartilage proteoglycans in the regenerated tissues from four specimens in the 25-mm group were detected by immunoblotting using specific monoclonal antibodies. Results: The operated limbs were subjected to an average of 80 to 90% pre-operative GRF after the eighth post-operative week and maintained till the end of the study. No significant difference was noted during the period between the three groups. A layer of regenerated tissue was noted on all specimens processed and was Sirius positive. Four operated specimens processed in the 25-mm group and three in the 28-mm group were Safranin O positive. The presence of cartilage aggrecan, cartilage link proteins, biglycan, and decorin was confirmed by immunoblotting. Discussion and Conclusion: We conclude that a cartilaginous resurfacing of acetabulum can be induced in vivo under the mechanical environment imposed by our hemi-arthroplasty model. This approach may be advantageous in clinical practice as a regenerated acetabular cartilaginous surface would avoid the problems associated with wear of the plastic acetabular cup and replacement of the acetabulum

Cultured primary cells have a limited life span and undergo dedifferentiation. Tissue engineering (TE) approaches require high cell numbers, but availability of human derived cells is limited and animal cells show inter-species differences. The advantages of immortalized cells are delayed senescence and phenotypic stability. The present study was undertaken to validate key properties of immortalized human anterior cruciate ligament (ACL) fibroblasts in direct comparison with non-immortalized cells from the same donor to assess their applicability as TE model. Human ACL ligamentocytes (40 years old female donor) were either immortalized using repeated transient transfection with a simian virus SV40 plasmid or remained untreated. Both cell populations were analyzed for cell survival, DNA content, tendon marker, extracellular matrix (ECM) and cytoskeletal protein expression. Cell spheroids of both populations were seeded on scaffolds embroidered either from polylactic acid (PLA) threads alone or combined PLA- and PLA-co-caprolacton-(P(LA-CL)) threads, functionalized with fluor treatment and collagen foams. Cell survival on the scaffolds was monitored for up to 5 weeks. In contrast to non-immortalized ligamentocytes, immortalized cells reflected some chaotic and incomplete cell divisions, higher DNA content, numbers of dying cells and nucleoli, reduced vimentin and vinculin-associated focal adhesions. Analysed markers, other cytoskeletal and ECM components were similarly expressed. Compared to the non-immortalized ligamentocytes immortalized formed instable spheroids and died on the scaffolds after 21 d. Both cell populations reflected superior growth on the PLA-P(LA-CL) compared with PLA scaffolds. Immortalized cells share crucial properties with their non-immortalized counterparts, but TE is only possible for limited culturing periods

Introduction: There is an ever-increasing clinical need for the regeneration and replacement of tissue to replace soft tissue lost due to trauma, disease and cosmetic surgery. A potential alternative to the current treatment modalities is the use of tissue engineering applications using mesenchymal stem cells that have been identified in many tissues including the fat pad. In this study, stem cells isolated from the fat pad were characterised and their differentiation potential assessed. Materials and Methods: The infrapatellar fat pad was obtained from total knee replacement for osteoarthritis. Cells were isolated, expanded and stained for a number of stem cell markers. For adipogenic differentiation, cells were cultured in adipogenic inducing medium (10ug/ml insulin, 1uM dexamthasone, 100uM indomethacin and 500uM 3-isobutyl-1-methyl xanthine). Gene expression analyses and Oil red O staining was performed to assess adipogenesis. Results: Cells at passage 2 stained strongly for CD13, CD29, CD44, CD90 and CD105 (mesenchymal stem cell markers). The cells stained sparsely for 3G5 (peri-cyte marker). On gene expression analyses, the cells cultured under adipogenic conditions had almost a 1,000 fold increase in expression of peroxisome proliferator-activated receptor gamma-2 (PPAR gamma-2) and 1,000,000 fold increase in expression of lipoprotein lipase (LPL). Oil red O staining revealed triglyceride accumulation within typical adipogenic morphology, confirming the adipogenic nature of the observed vacuoles, and showed failure of staining in control cells. Discussion: Fat pad derived stem cells expressed a cell surface epitope profile of mesenchymal stem cells, and exhibited the potential to undergo adipogenic differentiation. Our results show that the human fat pad is a viable potential autogeneic source for mesenchymal stem cells capable of adipogenic differentiation as well as previously documented ostegenic and chondrogenic differentiation. This cell source has potential use in tissue engineering applications

Tendons and tendon-to-bone entheses don't usually regenerate after injury, and the hierarchical organization of such tissues makes them challenging sites of study for tissue engineers. In this study, we have tried a novel approach using miRNA and a bioactive bioink to stimulate the regeneration of the enthesis. microRNAs (miRNAs) are short, non-coding sequences of RNA that act as post-transcriptional regulators of gene and protein expression [1]. Mimics or inhibitors of specific miRNAs can be used to restore lost functions at the cell level or improve healing at the tissue level [2,3]. We characterized the healing of a rat patellar enthesis and found that miRNA-16-5p was upregulated in the fibrotic portion of the injured tissue 10 days after the injury. Based on the reported interactions of miRNA-16-5p with the TGF-β pathway via targeting of SMAD3, we aimed to explore the effects of miRNA-16-5p mimics on the tenogenic differentiation of adipose-derived stem cells (ASCs) encapsulated in a bioactive bioink [4,5]. Bioinks with different properties are used for the 3D printing of biomimetic constructs. By integrating cells, materials, and bioactive molecules it is possible to tailor the regenerative capacity of the ink to meet the particular requirements of the tissue to engineer [5]. Here we have encapsulated ASCs in a gelatin-methacryloyl (GelMa) bioink that incorporates miR-16-5p mimics and magnetically responsive microfibers (MRFs). When the bioink is crosslinked in the presence of a magnetic field, the MRFs align unidirectionally to create an anisotropic construct with the ability to promote the tenogenic differentiation of the encapsulated ASCs. Additionally, the obtained GelMA hydrogels retained the encapsulated miRNA probes, which permitted the effective 3D transfection of the ASC and therefore, the regulation of gene expression, allowing to investigate the effects of the miR-16-5p mimics on the tenogenic differentiation of the ASCs in a biomimetic scenario.

The osteo-regenerative properties of allograft have recently been enhanced by addition of autogenous skeletal stem cells to treat orthopaedic conditions characterised by lost bone stock. There are however, multiple disadvantages to allograft, including cost, availability, consistency and potential for disease transmission, and trabecular tantalum represents a potential alternative. Tantalum is already in widespread orthopaedic use, although in applications where there is poor initial implant stability, or when tantalum is used in conjunction with bone grafting, loading may need to be limited until sound integration has occurred. Development of enhanced bone-implant integration strategies will improve patient outcomes, extending the clinical applications of tantalum as a substitute for allograft. The aim of this study was to examine the osteoconductive potential of trabecular tantalum in comparison to human allograft to determine its potential as an alternative to allograft. Human bone marrow stromal cells (500,000 cells per ml) were cultured on blocks of trabecular tantalum or allograft for 28 days in basal and osteogenic media. Molecular profiling, confocal and scanning electron microscopy, as well as live-dead staining and biochemical assays were used to characterise cell adherence, proliferation and phenotype. Cells displayed extensive adherence and proliferation throughout trabecular tantalum evidenced by CellTracker immunocytochemistry and SEM. Tantalum-cell constructs cultured in osteogenic conditions displayed extensive matrix production. Electron microscopy confirmed significant cellular growth through the tantalum to a depth of 5mm. In contrast to cells cultured with allograft in both basal and osteogenic conditions, cell proliferation assays showed significantly higher activity with tantalum than with allograft (P<0.01). Alkaline phosphatase (ALP) assay and molecular profiling confirmed no significant difference in expression of ALP, Runx-2, Col-1 and Sox-9 between cells cultured on tantalum and allograft. These studies demonstrate the ability of trabecular tantalum to support skeletal cell growth and osteogenic differentiation comparable to allograft. Trabecular tantalum represents a good alternative to allograft for tissue engineering osteo-regenerative strategies in the context of lost bone stock. Such clinical scenarios will become increasingly common given the ageing demographic, the projected rates of revision arthroplasty requiring bone stock replacement and the limitations of allograft. Further mechanical testing and in vivo studies are on-going

Background. Skeletal stem cells (SSCs) have been used for the treatment of osteonecrosis of the femoral head to prevent subsequent collapse. In isolation SSCs do not provide structural support but an innovative case series in Southampton, UK, has used SSCs in combination with impaction bone grafting (IBG) to improve both the biological and mechanical environment and to regenerate new bone at the necrotic site. Aims. Analysis of retrieved tissue-engineered bone as part of ongoing follow-up of this translational case series. Methods. With Proof-of-Concept established in vitro and in vivo, the use of a living bone composite of SSCs and allograft has been translated to four patients (five hips) for treatment of osteonecrosis of their femoral heads. Parallel in vitro culture of the implanted cell-graft construct was performed. Patient follow-up was by serial clinical and radiological examination. In one patient collapse occurred in both hips due to more advanced disease than was originally appreciated. This necessitated bilateral hip arthroplasty, but allowed retrieval of the femoral heads. These were analyzed for Type 1 Collagen production, bone morphology, bone density and mechanical strength by micro computed tomography (CT), histology (A/S stain, Collagen Type 1 immunostain, biorefringence) and mechanical testing. Representative sections of cortical, trabecular and tissue engineered bone were excised from the femoral heads using a diamond-tipped saw-blade and tested to failure by axial compression. Results. Parallel in vitro analysis demonstrated sustained cell growth and viability on the allograft. Three patients currently remain asymptomatic at up to three year follow-up. Histological analysis of the two retrieved femoral heads demonstrated, critically, Type 1 collagen production in the regenerated tissue as well as mature trabecular architecture, indicative of de novo tissue engineered bone. The trabecular morphology of regenerated bone was evident on CT, and this had a bone density of 1400 Grey scale units, (compared to 1200 for natural trabecular bone and 1800 for cortical bone). On axial compressive testing the regenerated bone on the left showed a 24.8% increase in compressive strength compared to ipsilateral normal trabecular bone, and a 22.9% increase on the left. Conclusions. Retrieval analysis data has demonstrated the translational potential of a living bone composite, while ongoing clinical follow-up shows this to be an effective new treatment for osteonecrosis of the femoral head. Regeneration of the necrotic bone may prevent subsequent collapse, thereby delaying, or possibly avoiding, the need for hip arthroplasty in early stage osteonecrosis. Evaluation of this tissue engineering construct has confirmed the potential for clinical treatment of bone defects using SSC based strategies

Angiogenesis and the ability to provide appropriate vascular supply are crucial for skeletal tissue engineering. The aim of this study was to investigate the angiogenic potential of human dental pulp stromal cells (HDPSCs) and stro-1 positive populations as well as their role in tissue regeneration (the clinical reality). HDPSC were isolated from the pulp tissues of human permanent teeth by collagenase digestion. STRO-1 positive cells were enriched using monoclonal anti- STRO-1 and anti- CD45 PE conjugated antibodies together with and fluorescence activated cell sorting (FACS). Cells isolated by FACS were grown to passage4 and cultured as monolayers or on 3D Matrigel scaffold in endothelial cell growth medium-2 (EGM-2) with/without 50ng/mL of vascular endothelial growth factor (VEGF). Cells cultured in alpha MEM supplemented with 10% FCS were used as controls. After 24, 48 and 72 hours angiogenic marker expression (CD31, CD34, vWF and VEGFR-2) was determined by qRT-PCR and immuno-histochemistry. Using three different donors, 0.5-1.5% of total HDPSCs population was characterized as STRO-1+/CD45- cells At each time point cells cultured as monolayer in EGM-2 with VEGF showed up regulation of CD31 and VEGFR-2 expression compared to the control group while expression of CD34 and vWF remained unaffected. However on Matrigel, all four genes were up regulated to different extents. CD31 and VEGFR-2 were up regulated to a greater degree compared to CD34 and vWF. Changes in gene expression in both cell types were time dependent. Immuno-histochemical staining confirmed that the HDPSCs cultured in the test group showed positive staining for the four angiogenic markers (CD31, CD34 vWF and VEGFR-2) when grown in both monolayer and 3D Matrigel culture compared to control cultures. When cultured on Matrigel (but not Monolayer) for 7 days, HDPSC formed tube-like structures in the VEGF treated group. This indicates the potential of use HDPSCs and their STRO-1 positive population for angiogenesis to enhance skeletal tissue repair and/or regeneration toward translational research for clinical benefit

Silk fibroin (SF) has been used as a scaffold for cartilage tissue engineering. Different silkworms strain produced different protein. Also, molecular weight of SF depends on extraction method. We hypothesised that strain of silkworm and method of SF extraction would effect biological properties of SF scaffold. Therefore, cell viability and chondrogenic gene expression of human chondrogenic progenitor cells (HCPCs) treated with SF from 10 silkworm strains and two common SF extraction methods were investigate in this study.

Twenty g of 10 strains silk cocoons were separately degummed in 0.02M Na2CO3 solution and dissolved in 100๐C for 30 minutes. Half of them were then dissolved in CaCl2/Ethanol/H2O [1:2:8 molar ratio] at 70±5๐C (method 1) and other half was dissolved in 46% w/v CaCl2 at 105±5๐C (method 2) for 4 hours. HCPCs were cultured in SF added cultured medial according to strain and extraction method. Cell viability at day 1, 3, and 7, were determined. Expression of collagen I, collagen II, and aggrecan at day 7 and 14, was studied. All experiment were done in triplicated samples.

Generally, method 1 SF extraction showed higher cell viability in all strains. Cell viability from Nanglai Saraburi, Laung Saraburi and Nangtui strains were higher than those without SF in every time point while Wanasawan and J108 had higher viability at day 1 and decreased by time. Expression in collagen 1, collagen 2 and aggrecan in method 1 are higher at day 7 and day 14. Collagen 1 expression was highest in Nangnoi Srisaket, followed by Laung Saraburi and Nanglai Saraburi in day 7. Nangnoi Srisaket also had highest expression at day 14, followed by Nanglai Saraburi and Laung Saraburi respectively. Nangseaw had highest collagen 2 expression, follow by Laung Saraburi and Nangnoi Srisaket respectively. Higher aggrecan gene expression of Tubtimsiam, Wanasawan, UB 1 and Nangnoi Srisaket was observed at day 7 and increased expression of all strains at day 14.

SF extraction using CaCl2/Ethanol/H2O offered better cell viability and chondrogenic expression. Nangseaw, Laung Saraburi and Nangnoi Srisaket strains expressed more chondrogenic phenotype.

Tissue engineering techniques, combining autologous chondrocytes with biodegradable biomaterials, may offer significant advantages over current articular cartilage repair strategies. We present a series of experiments investigating the effect of 3D scaffold architecture and biomaterial composition on cartilage tissue formation in vitro and in vivo. Porous polymer (PEGT/PBT) scaffolds with low (300/55/45) or high (1000/70/30) PEG molecular weight (MW) compositions were produced using novel solid free-form fabrication (3DF) techniques, allowing precise control over pore architecture, and conventional compression moulding (CM) foam techniques. Scaffolds were seeded with expanded human nasal chondrocytes, and cultured in vitro or implanted subcutaneously in vivo in nude mice for 4 weeks and cartilage tissue formation accessed. 3DF scaffolds contained highly accessible networks of large interconnecting pores (Ø525 μm) compared to CM scaffolds, containing complex networks of small interconnecting pores (Ø182 μm). 3DF scaffold architectures enhanced cell re-differentiation (GAG/DNA) and cartilaginous matrix accumulation compared to CM scaffolds, but only if 1000/70/30 compositions were used. Collagen type-II mRNA was significantly increased in 3DF architectures irrespective of scaffold composition. These effects were likely mediated by preferential protein adsorption to 1000/70/30 materials, promoting a spherical chondrocyte-like morphology, as well as efficient nutrient/waste exchange throughout interconnecting pores within 3DF architectures. We observed synergistic effects of both composition and 3D scaffold architecture on human chondrocyte re-differentiation capacity, however, our data suggests that scaffold composition has a more significant influence than architecture alone. Such design criteria could be included in future scaffold architectures for repairing articular cartilage defects

Purpose: Tissue engineering offers new therapeutic perspectives with the possibility of producing cartilage tissue for a large number of patients. These structures are composed of an absorbable synthetic support and competent cells. Two types of cells can be proposed: articular chondrocytes harvested from the peripheral part of the joint, or mesenchymatous stem cells (MSC) present in the bone marrow and possessing chondrogenic potential. The purpose of this study was to determine the optimal cell source and the best supporting material for in vitro production of cartilage. Material and methods: Isolated rabbit MSC were harvested and amplified with cell culture for 21 days. After this period, 20–40 million cells/ml were combined with polyglycolic acid sponges (3 types of sponges 1x1x0.2 cm2) and cultured in TGFß-enriched medium under specific dynamic conditions allowing gas exchange. The tissue obtained was compared with structures of identical size obtained with differentiated chondrocytes harvested from the same animals. The study included a histological analysis and immunohistochemistry for type I, II, and X collagen and biochemistry for DNA content, glycosaminoglycanes (GAG) and type II collagen. Results: After 3 weeks in culture, the composites obtained with MSC preserved their size and had the white pearly aspect of hyalin cartilage. The histological analysis and immunohistochemistry tests for type II collagen confirmed the presence of a cartilaginous matrix throughout the thickness of the fragments. The GAG and type II collagen contents were significantly higher with MSC compared with chondrocytes, irrespective of the supporting material. Discussion: This study demonstrated that cartilaginous tissue fragments can be obtained with MSC cultured on PGA supporting material under very specific conditions. Use of these cells offers the advantage of easy harvesting followed by in vitro amplification, and thus less harvesting morbidity. Complementary studies are needed to evaluate the behaviour of these living materials after implantation in the articulation

Human in vitro models of the neuromuscular junction (NMJ) are currently moving from embryonic stem cells to induced Pluripotent Stem Cells (iPSCs). With this, a robust model could be optimised for physiology and pathophysiology studies, as well as representing a drug screening platform. For this reason, the work presented here represents the optimisation of a human co-culture model of skeletal muscle (hSkM)/ iPSC-derived motor neurons (MNs) both in monolayer and in 3D tissue engineering collagen constructs. Firstly, human iPSC-derived motor neurons (MNs) were characterised over a period of 35 days to test their cholinergic potential. Then, primary human skeletal muscle (hSkM) and MNs were co-cultured on different substrates (gelatin and SureBond+ReadySet (Axol Bioscience)) and differentiated in various combinations of media to allow both myotube formation and neurite extension. Morphological (β-III Tubulin and Rhodamine Phalloidin) and interaction (α-Bungarotoxin and Synaptic Vesicle 2) immunofluorescent stainings were used to evaluate cell differentiation and co-localisation of pre and post-synaptic markers. Results from this study showed that the MNs presented a cholinergic phenotype up to 21 days; hSkM and MNs co-existed in culture and differentiated in neuronal Maintenance Medium (MM, Axol Bioscience); the 3D constructs allowed alignment and maturation of the muscle tissue, while providing a matrix for neurite extension and NMJ formation. This model has the potential to become a valid tool for in vitro drug screening while reducing the use of animals in research and providing the scientific community with a platform for personalised medicine

Cell sheets are manufactured from a high-density cell layer stabilized by its own freshly produced extracellular matrix (ECM). They could serve as versatile scaffolds for tissue repair. Unfortunately, their production often remains time-consuming requiring weeks of culturing. Ligament cell sheets are so far barely available. Regarding musculoskeletal tissues exposed to high repetitive biomechanical forces, the stability of cell sheets is insufficient. It could help to combine them with a biomechanical competent scaffold e.g. produced by an embroidering technique. Hence, we wanted to (1) develop a very rapid strategy to produce ACL ligamentocyte sheets within 24 h by using a thermoresponsive polymer surface, (2) use the sheets for scaffold seeding and (3) reflect the fibrocartilaginous transition zone of an ACL enthesis by combining sheets of ligamentocytes with chondrocytes or chondrogenic precursor cells as a strategy for directed seeding of two cell types on topologically different scaffold areas.

Different cell numbers of lapine ACL ligamentocytes (L-ACLs), lapine articular chondrocytes (L-ACs) and human mesenchymal stromal cells (H-MSCs) were used for sheet formation. Experiments were performed with novel, self-assembled poly(glycidyl ether) (PGE) brushes based on random glycidyl methyl ether and ethyl glycidyl ether copolymers on polystyrene 12-well cell culture plates, which allow rapid sheet formation within 24 h. Uncoated plates served as controls. Temperature-triggered detachment was performed by 10 min incubation with PBS at ambient temperature before treatment with fresh warm PBS for 5 min at 37 degrees Celsius. Harvested cell sheets were transferred on polyglycolic acid (PGA) or embroidered poly-lactic acid / poly-co-caprolactone (PLA/P[LA-CL]) scaffolds, functionalized with collagen foam and fluorine gas treatment (prepared at the IPF in Dresden and the FILK in Freiberg). Cell distribution, growth, vitality and synthesis of ECM components were monitored up to 7 days. Cell numbers required for sheet preparation (3.9 cm2) depended strongly on the cell type (L-ACLs: 0.395 mio/cm2, L-AC: 0.342 mio/cm2, H-MSCs: 0.131 mio/cm2) and was highest for L-ACLs. The majority of cells survived sheet assembly, detachment, transfer onto the scaffolds and culturing. Cells migrated from the sheets into the scaffolds and spread through the scaffolds. L-ACLs and L-ACs produced ECM and maintained their phenotypes (type II collagen and sulfated glycosaminoglycans in L-AC sheets, decorin and tenascin C in L-ACL sheets). The presence and distribution of two cell types in scaffold cocultures (L-ACLs and H-MSCs) was proven by anti-human vimentin labeling. Hence, the PGE brush surface allows rapid formation (24 h) of cell sheets.

In tissue engineering, scaffolds are vitalized by cells in vitro. Human mesenchymal stem cells (hMSC) are very interesting because of their ability to differentiate towards the osteogenic lineage and their self renewing capacity. Yet, it is important that implanted cells do not disseminate and exhibit unwanted cell growth outside the implantation site. Therefore the aim of this study was to detect migrated cells in organs of mice after implantation of a composite (cell-scaffold) substitute. HMSC (Cambrex, USA) were inoculated on a clinically approved 3D scaffold (Tutobone(TM), Tutogen, Germany). One composite and one scaffold without cells were implanted subcutanously, left and right paravertebrally in athymic nude mice (nu/nu). After 2, 4, 8 and 12 weeks constructs were explanted and organs (liver, spleen, lungs, kidney, heart, testicles, brain and blood) were harvested. The entire organs were homogenized and genomic DNA was isolated for qualitative and quantitative PCR. Human DNA was found in all explanted composites at all examined time points. No human DNA could be detected in control scaffolds. Moreover we did not detect human DNA in all explanted organs at any time point. As internal controls we could detect 1 single hMSC in a pool of 106 mouse cells. In conclusion, we could proof that cells of implanted composite substitutes do not migrate to other organs. Furthermore, this study showed that implanted hMSC seeded on 3D scaffolds survive over time frames up to 12 weeks

Dynamic compressive loading of cartilage can support extracellular matrix (ECM) synthesis whereas abnormal loading such as disuse, static loading or altered joint biomechanics can disrupt the ECM, suppress the biosynthetic activity of chondrocytes and lead to osteoarthritis. Interactions with the pericellular matrix are believed to play a critical role in the response of chondrocytes to mechanical signals. Loading of intact cartilage explants can stimulate proteoglycan synthesis immediately while the response of chondrocytes in tissue engineering constructs dependent on the day of culture. In order to effectively utilize mechanical signals in the clinic as a non-drug-based intervention to improve cartilage regeneration after surgical treatment, it is essential to understand how ECM accumulation influences the loading response. This study explored how construct maturity affects regulation of ECM synthesis of chondrocytes exposed to dynamic loading and unraveled the molecular correlates of this response. Human chondrocytes were expanded to passage 2, seeded into collagen scaffolds and cultured for 3, 21, or 35 days before exposure to a single loading episode. Dynamic compression was applied at 25% strain, 1 Hz, in 9 × 10 minute-intervals over 3h. Gene expression and protein alterations were characterized by qPCR and Western blotting. Proteoglycan and collagen synthesis were determined by radiolabel-incorporation over 24 hours. Maturation of constructs during culture significantly elevated ECM deposition according to histology and GAG/DNA content and chondrocytes redifferentiated as evident from raising COL2A1 and ACAN expression. Loading of d3 constructs significantly reduced proteoglycan synthesis and ACAN expression compared to controls while the identical loading episode stimulated GAG production significantly (1.45-fold, p=0.016) in day 35 constructs. Only in mature constructs, pERK1/2 and its immediate response gene FOS were stimulated by loading. Also, SOX9 protein increased after loading only in d21 and d35 but not in d3 constructs. Interestingly, levels of phosphorylated Smad 1/5/9 protein declined during construct maturation, but no evidence was obtained for load-induced changes in pSmad 1/5/9 although BMP2 and BMP6 expression were stimulated by loading. Selected MAPK-, calcium-, Wnt- and Notch-responsive genes raised significantly independent of construct maturity albeit with a generally weaker amplitude in d3 constructs. In conclusion, construct maturity determined whether cells showed an anabolic or catabolic response to the same loading episode and this was apparently determined by a differential SOX9 and pERK signaling response on a background of high versus low total pSmad1/5/9 protein levels. Next step is to use signaling inhibitors to investigate a causal relationship between Smad levels and a beneficial loading response in order to design cartilage replacement tissue for an optimal mechanical response for in vivo applications

INTRODUCTION. Bone marrow derived mesenchymal stem cells are a potential source of cells for the repair of articular cartilage defects. Hypoxia has been shown to improve chondrogenesis in adult stem cells. In this study we characterised bone marrow derived stem cells and investigated the effects of hypoxia on gene expression changes and chondrogenesis. MATERIALS AND METHODS. Adherent colony forming cells were isolated and cultured from the stromal component of bone marrow. The cells at passage 2 were characterised for stem cell surface epitopes, and then cultured as cell aggregates in chondrogenic medium under normoxic (20% oxygen) or hypoxic (5% oxygen) conditions for 14 days. Gene expression analysis, glycosoaminoglycan and DNA assays, and immunohistochemical staining were determined to assess chondrogenesis. RESULTS. Bone marrow derived adherent colony forming cells stained strongly for markers of adult mesenchymal stem cells including CD44, CD90 and CD105, and they were negative for the haematopoietic cell marker CD34 and for the neural and myogenic cell marker CD56. Interestingly, a high number of cells were also positive for the pericyte marker 3G5. Cell aggregates showed a chondrogenic response and in lowered oxygen there was increased matrix accumulation of proteoglycan, but less cell proliferation, which resulted in 3.2-fold more glycosoaminoglycan per DNA after 14 days of culture. In hypoxia there was increased expression of key transcription factor SOX6, and the expression of collagens II and XI, and aggrecan was also increased. DISCUSSION. Pericytes are a candidate stem cell in many tissue and our results show that bone marrow derived mesenchymal stem cells express the pericyte marker 3G5. The response to chondrogenic culture in these cells was enhanced by lowered oxygen tension, which up-regulated SOX6 and increased the synthesis and assembly of matrix during chondrogenesis. This has important implications for tissue engineering applications of bone marrow derived stem cells

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of time in culture. Biomaterial-based tendon implants are designed to withstand high physiological loads but often lack the appropriate biochemical, biophysical and biological structure to drive tendon regeneration by populating cells. The objective of this study is to use tendon main component, collagen type I, to create scaffolds that reproduce tendon natural anisotropy and rigidity, in an effort to engineer functional tendon tissue with native organization and strength, able to maintain tenocyte phenotype and to differentiate stem cells towards the tenogenic lineage. Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 um) or planar PDMS moulds and air-dried to obtain 5 mg/ml collagen films. Surface topography and elastic modulus were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were cultured on the micro-grooved/planar scaffolds for up to 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry. Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP (0, 0.5, 1 and 1.5mM) at low pH and temperature. The elastic modulus of the scaffolds cross-linked with 4SP (0.5mM) matched the values previously reported to induce tenogenic differentiation in stem cells (50–90 kPa). Approximately eighty percent of the human tendon cells cultured on the micro-grooved collagen films aligned in the direction of the anisotropy for 10 days in culture, mimicking the alignment of tenocytes in the native tissue. Cell nuclei morphology, known to play a central role in the process of mechanotransduction, was significantly more elongated for the tenocytes cultured on the micro-grooved scaffolds after 4 days in culture for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively on the micro-grooved and rigid scaffolds after 10 days in culture, respectively. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment. Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities.

Mesenchymal Stem Cells (MSCs) are key regulators in senile osteoporosis and in bone formation and regeneration. MSCs are therefore suitable candidates for stem cells mediated gene therapy of bone. Recombinant human Bone Morphogenetic Protein-2 (rhBMP-2) is a highly osteoinductive cytokine, promoting osteogenic differentiation of MSCs. We hypothesized that genetically engineered MSCs, expressing rhBMP2, can be utilized for targeted cell mediated gene therapy for local and systemic bone disorders and for bone/cartilage tissue engineering. Engineered MSCs expressing rhBMP-2 have both autocrine and paracrine effects enabling the engineered cells to actively participate in bone formation. We conditionally expressed rhBMP2 (tet-controlled gene expression, tet-off system) in mouse and human mesenchymal stem cells. RhBMP2 expressing clones (tet-off and adeno-BMP2 infected MSCs), spontaneously differentiated into osteogenic cells in vitro and in vivo. Engineered MSCs were transplanted locally and tracked in vivo in radial segmental defects (regenerating site) and in ectopic muscular and subcutaneous sites (non-regenerating sites). In vitro and in vivo analysis revealed rhBMP2 expression and function, confirmed by RT-PCR, ELISA, western blot, immunohistochemistry and bioassays. Secretion of rhBMP2 in vitro was controlled by tetracycline and resulted in secretion of 1231 ng/24 hours/106 cells. Quantitative Micro-CT 3-Dimentional reconstruction revealed complete bone regeneration regulated by tetracycline in vivo, indicating the potential of this platform for bone and cartilage tissue engineering. Angiogenesis, a crucial element in tissue engineering, was increased by 10-folds in transplants of rhBMP2 expressing MSCs (tet-off), shown by histomorphometry and MRI analysis (p< 0.05). In order to establish a gene therapy platform for systemic bone disorders, MSCs with tet-controlled rhBMP-2 expression, were injected systemically (iv). These engineered MSCs were genetically modified in order to achieve homing to the bone marrow. Systemic non invasive tracking of engineered MSCs was achieved by recording topographical bioluminescence derived from luciferase expression detected by a coupled charged CCD imaging camera. For clinical situations that require immuno-isolation of transplanted cells, we developed an additional platform utilizing cell encapsulation technique. Immuno-isolated engineered MSCs, with tet-controlled rhBMP-2 expression, encapsulated with sodium alginate induced bone formation by paracrine effect of secreted rhBMP-2. Finally, we have characterized a novel tissue-engineering platform composed of engineered MSCs and biodegradable polymeric scaffolds, creating a 3D bone tissue in rotating Bioreactors. Our results indicate that engineered MSCs and polymeric scaffolds can be utilized for ex vivo bone tissue engineering. We therefore conclude that genetically engineered MSCs expressing rhBMP-2 under tetracycline control are applicable for: a) local and systemic gene therapy to bone, and b) bone tissue engineering. Our studies should lead to the creation of gene therapy platforms for systemic and local bone diseases in humans and bone/cartilage tissue engineering

Purpose: Articular cartilage is a physiologically hypoxic tissue with a gradient of oxygen tension ranging from about 10% oxygen at the cartilage surface to less than 1% in the deepest layers. The overall goal of the study was to determine whether an injectable allogeneic/autologous fibrin scaffolds in combination with mesenchymal stem cells (MSCs) is suitable for articular cartilage tissue engineering, and to determine the effect of hypoxic culture conditions on the stability of cell-fibrin scaffolds. The secondary goal was to enhance the accumulation of extracellular matrix (ECM) inside the fibrin scaffold under these conditions. Method: Chondroprogenitor clonal cell line RCJ3.1C5.18 (C5.18) and human mesenchymal stem cells (hMSCs) were encapsulated in fibrin hydrogel and fibrin glue scaffolds. The stabilization of fibrin scaffolds and development of ECM components were evaluated using zymography, SDS-polyacrylamide electrophoresis (SDS-PAGE), immunochemistry, spectrophotometry, RT-PCR including real time and histology (. Ahmed TA., et al. . Tissue Engineering. 2007. ;. 13. (7): . 1469. –77. ). Results: After encapsulation of C5.18 and hMSCs, fibrin gels quickly degraded under normoxic conditions (21 % oxygen) due to upregulation of plasminogen and matrix metalloproteinases (MMPs) genes especially MMP-2, -3, and -9. Protease inhibitors such as aprotinin and galardin (GM6001), in combination or separately, prevented the fibrin-C5.18 hydrogels breakdown for up to 5 weeks. Only a combination of aprotinin and galardin resulted in accumulation of ECM components such as collagen II and aggrecan. In contrast, fibrin-hMSCs hydrogels were found to be stable under hypoxic conditions (5% O2) for up to 4 weeks in the absence of inhibitors, suggesting that hypoxic conditions may downregulate the expression of the enzymes responsible for fibrin-hydrogel breakdown. Conclusion: These results suggest that in C5.18 and MSCs cell lines, expression of matrix metalloproteinases (MMPs) and plasmin is upregulated under normoxic conditions and is responsible for fibrin-hydrogel breakdown. Moreover, inhibition of both proteases is required to enhance the accumulation of ECM. However, fibrin hydrogel scaffolds were stabilized under low oxygen tension, which is more physiological than normoxia and therefore these constructs may be stable after implantation in the absence of protease inhibitors

Bone tissue engineering has the intent to grow bone copies in the laboratory that could be used either for bone regeneration or as model systems to study bone physiology and pathology. Bone marrow- or adipose derived derived mesenchymal stromal cells are commonly used as they have been shown to be capable to differentiate into osteoblasts and depositing a calcium phosphate rich extracellular matrix. However, real bone is more than that: there are commonly three cell types described that are essential contributors to the tissue's native function: osteoblasts, osteocytes and osteoclasts. While all three cell types are being investigated separately, co-cultures of them including their precursors and inactive forms still provide a huge challenge these days, both in terms of culturing and (quantitative) evaluation. In addition, the matrix deposited by the osteoblasts in vitro is still far from bone's hierarchical organization in vivo that contributes to bone's impressive mechanical properties. Using a large set of microscopic tools (micro-computed tomography, SEM, 3D FIB/SEM, TEM and fluorescence), combined with spectroscopic (FTIR) and molecular tools (qPCR) we show that our 3D model system develops the main features of bone by human stromal cells differentiating first into osteoblasts who further embed themselves to become osteocytes. In their right environment and when stimulated mechanically, the cells are embedded within a collagenous matrix which is mineralized with carbonated hydroxyapatite. While this system still needs the addition of osteoclasts to represent ‘real’ bone, it allows to study the interaction between osteoblasts and osteocytes and to invest parameters contributing to collagen mineralization in high resolution and cryogenic conditions.

The potential of piezoelectric biomaterials for bone tissue engineering is demonstrated. This work proves that the use of piezoelectric poly(vinylidene fluoride) (PVDF), able to provide electrical stimuli upon mechanical solicitation to the growing bone cells, enhances the bone regeneration in vivo. Poled and non-poled PVDF films, with and without macroscopic piezoelectric response, respectively and randomly oriented piezoelectric electrospun fiber mats have been used as substitutes for bone to test their osteogenic properties in Wistar rats by analyzing new bone formation in 3 mm bilateral femur defects in vivo. After 4 weeks, the qualification of the regenerated bone was performed according the H&E staining. Defect implanted with poled PVDF films demonstrated significantly more defect closure and bone remodeling, showing the large potential of piezoelectric biomaterials for bone repair, as well as for other electromechanical responsive tissues such as muscle and tendon.

To repair soft tissue, it is vital to ensure that the biomaterial is able to mimic the complex elasticity of the native tissue. It has been demonstrated that substrate stiffness has a huge influence on cellular growth, differentiation, motility and phenotype maintenance. The goal of the present study is to characterize extensively a set of polymeric films with variable mechanical profiles. A range of synthetic biodegradable polymers was selected according to the physico-chemical intrinsic properties of aliphatic polymers. They have similar chemistry (absorbable polyesters made from lactic acid, glycolic acid, trimethylene carbonate, dioxanone & β-caprolactone), however show different mechanical and degradation properties. The films were manufactured by thermal presser and then characterized by scanning electron microscopy (SEM), differential scanning calorimetry (DSC), nuclear magnetic resonance spectroscopy (NMR) and Fourier transform infrared spectroscopy (FTIR). The mechanical properties of the films were assessed by uniaxial tensile tests in wet conditions and also by atomic force microscopy (AFM) to assess the material's stiffness at a micro-level. In vitro assays were performed to assess the cell cytocompatibility, proliferation and differentiation potential of the films. The mechanical properties of the materials are within the range intended for musculoskeletal tissue repair. Biological assays showed good cell adhesion, cell proliferation and cell viability. Stem cells were able to differentiate into adipogenic, osteogenic, chondrogenic and tenogenic lineages. Overall the selection of polymers gave good options for a potential tissue repair scaffold. In the future, the combined effect of stiffness and topography will be assessed on cell phenotype maintenance.

Significant challenges remain to accomplishing the development of fully functional tendon tissue substitutes that can lead to clinically effective and successful applications. Scaffolding materials must meet demanding requirements such i) mimic the hierarchical and anisotropically aligned structure of tendon tissues from the nano- up to the macroscale, ii) meet tendon mechanical requirements and non-linear biomechanical behaviour, iii) provide the necessary biophysical/biochemical cues and mechanical responsiveness to induce the tenogenic differentiation of stem cells and potentiating the effects of biochemical supplementation. On the other side, tenogenic differentiation of stem cells is still to be established, as well as the role of such cells (either naïve or pre-differentiated) in promoting tissue regeneration. We have recently found evidences that magnetic actuation can provide means of mechanically stimulating cells in a contact-free manner and, more interestingly, can also modulate inflammatory response, a critical issue for achieving tissue regeneration instead of repair. In summary, synergies of scaffold design and magnetic responsiveness can impact significantly cells behaviour as well as in vivo response and thus widen the therapeutically range of cell-laden tissue engineered constructs in tendon regeneration.

Adherent cells are known to respond to physical characteristics of their surrounding microenvironment, adapting their cytoskeleton and initiating signaling cascades specific to the type of cue encountered. Scaffolds mimicking native biophysical cues have proven to differentiate stem cells towards tissue-specific lineages and to maintain the phenotype of somatic cells for longer periods of culture time. Although the characteristic anisotropy of tendon tissue is commonly replicated in scaffolds, relevant physical cues such as tendon rigidity or mechanical loading are often neglected. The objective of this study is to use tendons' main extracellular matrix component, collagen type I, to create scaffolds with an anisotropic surface topography and controlled rigidity, in an effort to engineer functional tendon tissue equivalents, with native organization and strength.

Porcine collagen type I in solution was treated with one of the following cross-linkers: glutaraldehyde, genipin or 4-arm polyethylene glycol (4SP). The resulting mixture was poured on micro-grooved (2×2×2 μm) or planar polydimethylsiloxane (PDMS) molds and dried in a laminar flow hood to obtain 5 mg/ml collagen films. Surface topography and elastic modulus of the final scaffolds were analyzed using SEM/AFM and rheometry, respectively. Human tendon cells were isolated from adult tendon tissue and cultured on micro-grooved/planar scaffolds for 4, 7 and 10 days. Cell morphology, collagen III and tenascin C expression were analyzed by immunocytochemistry.

Among the different cross-linkers used, only the treatment with 4SP resulted in scaffolds with a recognizable micro-grooved surface topography. Precise control over the micro-grooved topography and the rigidity of the scaffolds was achieved by cross-linking the collagen with varying concentrations of 4SP at low pH and temperature. The elastic modulus of the scaffolds cross-linked with the highest concentration of 4SP matched the physiological values reported in developing tendons (∼15 kPa). Around eighty percent of the human tendon cells cultured on the cross-linked collagen films aligned in the direction of the anisotropy for 10 days in culture. At 4 days, tenoyctes cultured on micro-grooved substrates presented a significant higher nuclei aspect ratio than tenocytes cultured on planar substrates for all the 4SP concentrations. Synthesis, deposition and alignment of collagen III and tenascin C, two important tenogenic markers, were up regulated selectively in the rigid micro-grooved scaffolds after 7 days in culture. These results highlight the synergistic effect of matrix rigidity and cell alignment on tenogenic cell lineage commitment.

Collectively, this study provides new insights into how collagen can be modulated to create scaffolds with precise imprinted topographies and controlled rigidities. Gene expression analysis and a replicate study with hBMSCs will be carried out to support the first results and to further identify the optimal biophysical conditions for tenogenic cell lineage commitment. This potentially leads to the design of smart implants that not only restore immediate tendon functionality but also provide microscopic cues that drive cellular synthesis of organized tissue-specific matrix.

Objective: Meniscus regeneration is limited, moreover, loss of meniscal tissue leads to osteoarthritis. A new biomaterial, consisting of hyaluronic acid and polycaprolactone was applied as a meniscus substitute device in a study in sheep.

Methods: 24 sheep received a total medial meniscal replacement. Group SCF (n=12) was treated with a cell free scaffold, Group SCS (n=12) with a scaffold seeded with autologous chondrocytes harvested from the contralateral joints, which served as sham controls (n=12). Further 12 non-operated and 2 menisectomy controls were included in the study.

The animals were sacrificed after 4 months. The implants and joint surfaces were evaluated on a macroscopic (Implant Gross Assessment Score; Gross Assessment of Joints Score) and histological level.

Results: There was no significant difference of the Implant Gross Assessment score between the SCF and SCS groups. All implants showed excellent capsular ingrowth at the periphery. Dislocation, extrusion and tears occurred in part of the specimen due to biomechanical problems caused by soft tissue quality. The mean Gross Assessment of Joint Changes Score of the groups SCF and SCS was not statistically different. Cartilage damage was significantly more severe in joints with implants than in non-operated joints and sham controls, but less pronounced than in menisectomy controls. Histological evaluation showed residual scaffold with an associated foreign body response in all implants. Fibrous tissue was present in all implants, in contrast small foci of cartilaginous differentiation were more common in the cell-seeded constructs.

Discussion: At 4 months regenerative meniscal tissue was present but immature. The present study showed that strong biomechanical scaffold properties are a required to allow guided tissue regeneration and maturation under loading conditions. Cell seeding of the scaffold encourages cartilaginous differentiation. Modification of the scaffold and the cell-seeding technique will be investigated in further studies.

Introduction: Total meniscectomy can cause cartilage degeneration and osteoarthritis. The healing capacity of the meniscus is limited. Bioengineered meniscus can be a valid therapeutic option. Within the framework of the European Project MENISCUS, a pilot animal study was conducted to evaluate surgical technique, critical defect size, implant ingrowth and postoperative mobilization using a meniscus replacement device.

Materials and Method: Six sheep were operated on their right stifle joints. 3 sheep received a total meniscus replacement with a 3D biomaterial fixed with sutures. Additionally, controls without implant were operated. The sheep were sacrificed and evaluated clinically and histologically after 6 weeks.

Results: All implants showed excellent adhesion to the capsule and a good ingrowth at the periphery and the horns. Tissue formation was confirmed histologically.

Conclusions: Tissue ingrowth of the implant was demonstrated. The promising results concerning tissue formation and its meniscus like properties will have to be confirmed in future long-term studies.

Introduction

Articular hyaline cartilage has a unique structural composition that allows it to endure high load, distribute load to bone and enables low friction movement in joints. A novel acellular xenogenic graft is proposed as a biological cartilage replacement, for repair of osteochondral defects. Acellular porcine cartilage has been produced using repeated freeze thaw cycles and washing using hypotonic buffers and sodium dodecyl sulphate solution (SDS; Keir, 2008). DNA content of the acellular matrix was reduced by 93.3% compared to native cartilage as measured by nanodrop spectrophotometry of extracted DNA, with a corresponding reduction in glycosaminoglycan (GAG) content.

Autologous chondrocyte transplantation has become a possible solution for the treatment of chondral knee lesions. Recently an autologous tissue engineered cartilage (Hyalograft C), using biodegradable scaffolds for cell proliferation, was successfully developed. In osteochondritis dissecans (OCD) the lesion also involves subchondral bone. For this reason we began to use a two-step technique: arthroscopic autologous bone grafting followed by autologouos condrocyte Hyalograft C transplantation after 4–6 months. We treated five patients affected by OCD.

All the patients were clinically evaluated and analysed according to the International Repair Cartilage Society score at 12 and 24 months. The ICRS score showed highly satisfactory clinical results in all treated patients at 12 and 24 months; CT and MRI evaluation had demonstrated a good articular surface reconstruction with complete bone defect restoration at a short 12-month follow-up period. The autologous chondrocyte transplantation provides highly satisfactory clinical results.

This second-generation autologous tissue-engineered cartilage transplantation avoids the use of periosteal flap, simplifies the surgical procedure and permits use of an arthroscopic approach. In association with autologous bone grafting, bone loss can also be restored in order to recreate a perfect articular surface. The preliminary clinical and histological results are encouraging but longer follow-up is required to better evaluate this technique.

A tissue engineering-based approach has become a possible solution for the treatment of chondral lesions. Actually, autologous chondrocytes seeded on biodegradable scaffolds for cell proliferation were successfully developed. However, these techniques promote cartilaginous but not bony regeneration. Therefore a new experimental approach involving mesenchymal stem cells (MSC) has been introduced.

A 31-year-old man affected by massive osteonecrosis of the right femoral head was selected to begin this study. The MSC were isolated from the bone marrow harvested from the patient’s iliac crest. After a 3-week monolayer expansion, cells were seeded and cultured onto hyaluronan-based three-dimensional scaffolds and DBM spongy chips, used to regenerate the cartilaginous and the bony portion, respectively. After a 2-week cultivation, constructs were implanted inside the osteochondral defect using the transtrochanteric approach under arthroscopic control. The patient underwent clinical, X-ray and MRI control during the first 6 months after operation.

Pluripotent MSC may be a promising strategy for osteochondral defect reconstruction due to their capacity to differentiate in vivo along chondrocytic and osteoblastic lineages. This ability, combined with two different kinds of three-dimensional scaffolds, permits simultaneous bone and cartilage tissue regeneration. The preliminary results are encouraging but a more precise judgement of the effectiveness of this method requires longer follow-up.

Collagen scaffolds are generally characterized by their random fibre distribution and weak mechanical properties, which makes them unsuitable as substitutes for highly anisotropic tissues such as cornea or tendon. Recently, we developed a technique to create collagen type I scaffolds with well-defined anisotropic micro-patterns. Porcine collagen was mixed with PBS10X, NaOH and one of the following cross-linkers: glutaraldehyde (GTA), genipin and 4-arm polyethylene glycol (4SP). The resulting mixture was casted on micro-grooved (2×2×2 μm) polydimethylsiloxane (PDMS) moulds and allowed to dry in a laminar flow hood to obtain 5mg/ml collagen films. Different pH, temperatures (Tº), and cross-linker concentrations were tested in the process. Collagen gelation kinetics was analysed with rheometry and surface topography was assessed with scanning electron microscopy (SEM). Human bone marrow stem cells (HBMSCs) were seeded on the films and cell alignment was analysed by rhodamine/phalloidin staining and imaged with fluorescence microscopy. From all three cross-linkers tested, only 4SP cross-linked scaffolds showed a well-defined micro-grooved pattern. Increasing pH and Tº on 4SP-treated collagen decreased gelation time, which resulted in complete inhibition of the pattern, suggesting that an initial low viscous solution is required for a correct PDMS pattern infiltration. A wide range of 4SP concentrations (0.5, 1, 1.5 mM) maintained the well-defined topography on the films, opening the door to future fine-tuning of the stiffness sensed by cells. hBMSCs seeded on top of the scaffolds aligned along the pattern for 14 days in culture. Collectively, this data highlights the potential of these collagen scaffolds as tendon substitutes.

Composite tissue defects in absence of general contra-indications are now routinely dealt by composite free tissue transfers which now stand as routine procedures. When dealing with amputations of the thumb several reconstructive procedures are now available and should be tailored on individual basis.

Microvascular surgical techniques are especially gratifying when the appropriate indications are followed: young age, absence of systemic diseases no alcohol or smoke abuse.

A custom – made thumb can be reconstructed assembling in a single composite tissue graft different tissues harvested from various parts of the body.

An outcome study as been performed on 72 patients where the wrap-around technique, the second and great toe transfer have been carried out.

Overall assessment included, as a preliminary analysis, the of vassessement behaviour of each single transferred tissue: the nail complex, the pulp and the bone. Donor – site problems at the foot were assessed by gait analysis. Vascular complications were recorded in 5% of the patients, and 89% of the patients had excellent outcomes.

The formation of biomimetic environments using scaffolds containing cell recognition sequence and osteo-inductive factors in combination with bone cells offers tremendous potential for bone and cartilage regeneration. In tissues, collagen forms the scaffold by mediating the flux of chemical and mechanical stimuli. Recently, a synthetic 15-residue peptide P-15, related biologically to the active domain of type I collagen, has been found to promote attachment and the osteoblast phenotype of human dermal fibroblasts and periodontal ligament fibroblasts on particulate anorganic bone mineral (ABM). The aim of this study was to exam the ability of the collagen peptide, P-15, to promote human osteoprogenitor attachment, proliferation and differentiation on cell culture surfaces and 3-D scaffolds.

Selected human bone marrow cells were cultured on particulate microporous anorganic bone mineral (‘pure ‘ hydroxyapatite based on x-ray diffraction standard JCPDS9-432) phase and polygalactin vicryl mesh adsorbed with or without P-15 in basal or osteogenic conditions. Cell adhesion, spreading and patterning were examined by light and confocal microscopy following incorporation of cell tracker green and ethidium homodimer fluorescent labels. Osteoprogenitor proliferation and differentiation was assessed by DNA content and alkaline phosphatase specific activity. Growth and differentiation on 3-D ABM structures were examined by confocal and scanning electron microscopy (SEM).

P-15 promoted human osteoprogenitor cell attachment and patterning on particulate bovine anorganic bone mineral phase and polygalactin vicryl mesh over 5–24 hours compared to culture on ABM and vicryl mesh alone as observed by photomicroscopy. Increased alkaline phosphatase specific activity was enhanced following culture on P-15 adsorbed matrices as recognized by enhanced expression of alkaline phosphatase, type I collagen, osteocalcin and cfba-1. The presence of mineralised bone matrix and extensive cell ingrowth and cellular bridging between 3-D ABM matrices and polygalactin vicryl mesh adsorbed with P-15 was observed by confocal microscopy and alizarin red staining. SEM confirmed the 3-D structure of newly formed cell constructs and cellular ingrowth on and between the P-15 modified inorganic bone mineral materials. Negligible cell growth was observed on ABM alone or polygalactin vicryl mesh alone.

These observations demonstrate that the synthetic 15-residue collagen peptide, P-15, when adsorbed to ABM or polygalactin vicryl mesh, can stimulate human osteoprogenitor attachment and spreading. They also demonstrated that P-15 coupled 3-D matrices stimulate human osteoprogenitor differentiation and materialisation. The studies indicate that a synthetic analogue of collagen provides a biomimetic environment supportive for cell differentiation and tissue regeneration and indicate a potential for the use of extracellular matrix cue in the development of biomimetic environments for bone tissue engineering.

In this study, a biomimetic triphasic scaffold was constructed to mimic the native cartilage-subchondral bone tissue structure. This scaffold contained chondral layer, calcified zone of cartilage (CZC) and subchondral bone layer. The chondral layer was type II collagen sponge, the CZC and the subchondral bone layer were derived from normal pig knee by decellularization. In order to build separate microenvironment for chondral layer and subchondral bone layer, a dual-chamber bioreactor was designed by computer aided design, manufactured by 3D printer using Poly Lactic Acid, with CZC as the barrier of these two chambers. Culture medium in these two chambers was circulated separately by peristaltic pumps. Amniotic mesenchymal stem cells were seeded in this scaffold, fluorescence labeling was used for cell tracking, total DNA content analysis was used to indicate cell proliferation, and inducing medium was used to direct stem cells differentiation. After 7 days culture, the cells regularly distributed in the scaffold, cell adhesion and proliferation was not affected. No cell migration across CZC occurred. Total DNA content analysis showed that cells in scaffold increased in a time-dependent manner. Chondrogenic and osteogenic medium could induce stem cells in these two chambers to differentiate into chondrocytes and osteocytes, respectively. Our pilot study showed that the dual-chamber culture system with biomimetic triphasic scaffold was feasible, therefore this system will be further modified and tested in vivo.

Perivascular stem cells (PSCs) from lipoaspirate demonstrate increased purity and immaturity with greater engraftment potential than standard mesenchymal stem cells (MSCs). MSCs from the infra-patellar fat pad (IFP) have previously demonstrated increased chondrogenic potential. This study investigated the availability and potential of PSCs harvested from the infra-patellar fat pad of the human knee for musculoskeletal regeneration.

Tissue sections of IFP were stained with markers for PSCs, MSCs and endothelial cells to confirm their presence and location. Samples were obtained from patients undergoing TKR (n=13) or ACL reconstructions (n=10). Pericytes and adventitial cells made up 3.8% and 21.2% respectively of the stromal vascular fraction. The total number of pericytes and adventitial cells were 4.6±2.2×104 and 16.2±3.2×104 respectively. Cells were cultured both separately and combined. Cell identity was ascertained using fluorescence-activated cell sorting, immunocytochemistry and PCR. Cultured PSCs were differentiated using chondrogneic, osteogenic, adipogenic and myogenic medias. Differentiation was determined using Alcian Blue, Alizarin red, Oil Red O and myosin staining.

This study demonstrates that the IPFP is a viable source of PSCs that can be harvested either arthroscopically or through an arthrotomy by orthopaedic surgeons for cell-based musculoskeletal regeneration. Their potential now needs to be compared to conventional MSCs.

Perivascular stem cells (PSCs) from lipoaspirate demonstrate increased purity and immaturity with greater engraftment potential than standard mesenchymal stem cells (MSCs). MSCs from the infra-patellar fat pad (IFP) have previously demonstrated increased chondrogenic potential. This study investigated the availability and potential of PSCs harvested from the infra-patellar fat pad of the human knee for musculoskeletal regeneration.

Sections of IFP were stained with markers for PSCs, MSCs and endothelial cells to confirm their presence and location. Samples were obtained from patients undergoing TKR (n=13) or ACL reconstructions (n=10). Pericytes and adventitial cells made up 3.8% and 21.2% respectively of the stromal vascular fraction. The total number of pericytes and adventitial cells were 4.6±2.2×10⁴ and 16.2±3.2×10⁴ respectively. Cells were cultured both separately and combined. Cell identity was ascertained using fluorescence-activated cell sorting and immunocytochemistry. Cultured PSCs were differentiated using chondrogneic, osteogenic, adipogenic and myogenic medias. Differentiation was determined using Alcian Blue, Alizarin red, Oil Red O and mysosin staining.

This study demonstrates that the IFP is a viable source of PSCs that can be harvested either arthroscopically or through an arthrotomy by orthopaedic surgeons for cell-based musculoskeletal regeneration. Their potential now needs to be compared to conventional MSCs.

Aim of the study: To evaluate the use of a gelfoam sponge as a scaffold material in delivering osteoblast cells transfected with the VEGF gene for fracture repair.

Methods: In vitro: Osteoblasts were cultured from periosteum of rabbit bone and labeled with the visible CMTMR. Commercially available gelfoam with 12 pieces (each 3 × 3 × 3 mm3) was impregnated and cultured with the labelled cells (1×106) in a 12 wells plate for 1, 3 and 7 days. We embedded the gelfoam with labeled cells in an OCT compound enface, and the sections were then examined under a fluorescent microscope. In vivo: Osteoblasts were transfected with VEGF by use of SuperFect (Qiagen Inc) and cultured for 24 hours. The gelfoam pieces were impregnated with the transfected cells (5×106) saline solution for 30 minutes and placed into a segmental bone defect created in the rabbit tibia for 7 (n=3) and 14 (n=3) days. The specimens including the new bone were cut through each site of the segmental defect and embedded in paraffin. The sections were dewaxed and immunostained with mouse anti-human VEGF.

Results: In vitro: CMTMR-labeled cells survived and were detected within gelfoam at different time intervals (days 1, 3 and 7). In vivo: Immunostained VEGF proteins were visualized in the tissues surrounding the residual gel-foam at the fracture site at days 7 and 14 post surgery.

Conclusion: Our results indicate that the labeled/transfected cells are capable of growth in a gelfoam sponge both in vitro and in vivo.

The ability to generate replacement human tissues on demand is a major clinical need. Indeed the paucity of techniques in reconstructive surgery and trauma emphasize the urgent requirement for alternative strategies for the formation of new tissues and organs. The idea of biomimesis is to abstract good design principles and optimizations from nature and incorporate them in the construction of synthetic materials and structures. Direct appropriation of natural inorganic skeletons is also biomimetic since their unique properties inform us on ways to generate functional, optimized scaffolds.

A number of well characterized natural skeletons were investigated as potential scaffolds for tissue regeneration using mesenchymal stem cell populations. Marine sponges, sea urchin skeletons and nacre were found to possess unique functional properties that supported human cell attachment, growth and proliferation and provided organic/ inorganic extracellular matrix analogues for guided tissue regeneration.

A good understanding of the processses involved in biomineralisation and the emergence of complex inorganic forms has inspired synthetic strategies for the formation of biological analogues (organised inorganic materials with biological form). We have developed two functional examples of biological structures generated using biomimetic materials chemistry with applications for human tissue regeneration. Mineralised biopoly-saccharide microcapsules provided enclosed micro-environments with an appropriate physical structure and physiological milieu, for the support of the initial stages of tissue regeneration combined with a capacity to deliver human cells, plasmid DNA and controlled release of biological factors such as cytokines. Calcium carbonate porous microspheres analogous to microscopic coccolithophore shells provided a template for tissue formation and a mechanism for the delivery of DNA and functional biological factors. These biomi-metic structures have considerable potential as scaffolds for skeletal repair and regeneration, particularly when combined with inductive and stimulatory biological factors (cytokines, morphogens, signal molecules) and plasmid DNA carrying with them chemical cues that modulate and direct permanent tissue formation complimentary with the host.

Purpose: The objective of this project is to determine the suitability of modified fibrin hydrogels as scaffolds for articular cartilage tissue engineering. The attractive feature of the fibrin system is that the gel precursors are available in autologous form. We have previously demonstrated that genipin, a naturally occurring cross-linking agent, stabilizes the fibrin gel.

Methods: Human articular chondrocytes were isolated from articular cartilage harvested from consenting patients undergoing total knee arthroplasty. The human cells were encapsulated into fibrin gels where gelation was induced by combining fibrinogen, thrombin, and genipin. The resulting gels were evaluated for extracellular matrix (ECM) production, mechanical properties, cell viability, and biodegradation.

Results: No breakdown of the gels was detected during 5 weeks of cell culture. After several weeks in culture, histology indicates significant proteoglycan production by encapsulated cells, and collagen II and aggrecan were detected in this ECM by immunostaining. There was a greater accumulation of cartilage-like ECM in the gels cross-linked with genipin. Dynamic compression tests performed at 0.1 Hz for 10 cycles using an MTS machine indicate that accumulation of ECM was associated with increased stiffness of the material. Cell viability was assessed using live/dead staining, and was found to be > 50% after 24 hours and at 1 week in culture. The presence of genipin cross-linking did not significantly affect cell viability. Real-Time RT-PCR indicated that encapsulated chondrocytes show an increase in Sox9, collagen II and aggrecan expression over 5 weeks and that this is further increased in the presence of genipin. The gene expression results agreed with the enhanced ECM seen under these conditions by histology and immunostaining. The fibrin material was also implanted subcutaneously into rats and after 30 days the material was removed, sectioned and evaluated. Immunostaining indicated that while there was evidence of biodegradation, the material did not appear to cause an inflammatory response.

Conclusions: Modified fibrin hydrogels show potential as cellular scaffolds for articular cartilage tissue engineering. An in vivo orthopaedic model must now be developed to fully evaluate the potential of the fibrin gel. Funding: Other Education Grant

Introduction: The aim of this study was to develop a technique to decellularise a porcine cartilagebone construct with a view to using this as a biological scaffold for transplantation into human osteochondral defect as a cartilage substitute.

Methods: Decellularisation was based on a modification of the technique of Booth et al (2002). Cartilage bone matrix (n=9) were decellularised by exposing the tissue to 2 cycles of dry freeze-thaw followed 2 more cycles with the addition of hypotonic (10mM tris-HCl, pH8.0) buffer. Samples were then cycled through hypotonic buffer, followed by ionic detergent (0.1% [w/v] sodium dodecyl sulphate [SDS]) in the presence of protease inhibitors (aprotinin 10 KIU/ml) and 0.1% (w/v) ethylene diamine tetraacetic acid (EDTA). This was followed by washes in PBS with aprotinin and incubation in nuclease solution containing DNase (50U/ml) and RNase (1U/ml). Decontamination using 0.1% (v/v) peracetic acid in PBS was then incorporated to achieve disinfection of the tissue samples. Finally, samples were washed in PBS. Three decellularisation protocols were used depending on the number of hypotonic/SDS cycles: this was either done once, three or six times referred to as DC1, DC3 and DC6 respectively. Fresh & decellularised cartilage were compared histologically using haematoxylin and eosin staining, to visualize cellular content, sirius red, to visualise collagen fibres & alcian blue, to visualise glycosaminoglycans (GAG). Immunohistochemistry staining for galactose-α-1,3-galactose (α-gal), collagen I, II & VI was performed for fresh and decellularised samples. DNA assay: Genomic DNA was extracted using a DNA isolation kit for tissues (Roche Applied Sciences). Collagen and DMB sulphated sugar assay, as described by Stapleton et al. (2008), were performed to measure collagen and GAG content. The biphasic property of fresh and decellularised cartilage was determined using a pin on plate indentation test.

Results: H& E staining revealed the absence of visible whole cells. Sirius red stain gave evidence of the retention of collagen following decellularisation. In contrast, the acellular matrix showed evidence of loss of GAGs. There was no evidence of the expression of α-gal in the acellular scaffold. DNA analysis revealed the absence of genomic DNA in comparison to fresh tissues (ANOVA, p< 0.05). The decellularisation process had minimal effect on the collagen content of the cartilage. Nevertheless there was a significant difference in the sulphated sugar content of the fresh tissue when compared to the decellularised tissue (ANOVA, p< 0.05), indicating loss of 92% GAG. Biomechanical testing of decellularised tissues showed a significant change (ANOVA, p< 0.05) in comparison to the fresh cartilage.

Discussion: In conclusion this study has generated data on the production of an acellular cartilage bone matrix scaffold for use in osteochondral defect repair. To our knowledge, this is the first study that has successfully removed whole cells and α-gal from xenogeneic cartilage and bone tissue. Future studies are required to investigate methods to recellularise the acellular matrix using an appropriate cell type and mechanical conditioning and to investigate replenishing GAG loss following decellularisation.

Introduction: This study tested the hypothesis that the use of biomaterials in distraction osteogenesis (DO) would reduce the treatment time and enhance bone formation in bone defect management.

Methods: A 1.0cm tibial shaft was removed in the left tibia of 36 rabbits. Rabbits were divided into three groups: Group A, the defect gap was reduced with the tibia shortened for 1.0-cm; Group B, the defect gap was filled with 1.0-cm porous hydroxyapatite/tri-calcium phosphates cylindrical block (HA/TCP block, diameter 0.5-cm); Group C, The 1.0-cm defect gap was reduced 0.5cm and the remaining 0.5-cm defect gap was filled with 0.5-cm HA/TCP block. The tibia was then fixed with unilateral lengthener; for groups A and C, lengthening started 7 days after surgery at a rate of 1.0 mm/day, in two steps. Group A received lengthening for 10 days and Group C for 5 days, there was no lengthening for Group B. All animals were terminated at day 37 following surgery. The excised bone specimens were subject to micro-CT, mechanical testing and histological examinations.

Results: Bone mineral density and content and tissue mineral density and content, as well as the mechanical properties of the regenerates were significantly higher in Group C compared to Groups A and B. Micro CT and histological examinations also confirmed that the regenerates in Group C had most advanced bone formation, consolidation and remodeling compared to other groups.

Conclusion: The combined use of biomaterials and DO technique can reduce the treatment time and enhance bone consolidation in bone defect management.

Flock technology is well known from textile industry. Short fibres are applied vertically on a substrate, coated with a flocking adhesive. Until now this technology has not been used in the field of biomaterials although it offers the possibility to create anisotrophic matrices with a high compressive strength despite of high porosity. Matrices presently used in matrix assisted autologous chondrocyte implantation do not show any orientation of the embedded chondrocytes. However column orientation and anisotropic direction of embedded cells and collagen fibers are thought to be necessary for proper cartilage matrix biomechanics. Combination of matrices as a guiding structure and chondrogenically differentiated mesenchymal stem cells (MSC) could offer new possibilities in the treatment of cartilage defects. Our aim was to evaluate whether anisotropic scaffolds are capable to support a cellular cartilaginous phenotype in vitro.

Electrostatically flocked matrices consisted of a collagen substrate, gelatine as adhesive and polyamide flock fibres. Chondrogenic cells and MSC were embedded in the scaffolds. Adherence, vitality and proliferation was assessed using confocal laser-scan microscopy (cLSM). Chondrogenic induction was performed in the presence of TGF-beta 3. Accumulation of proteoglycans was quantified by alcian-blue stain and collagen type II synthesis after extraction of the newly synthesized matrix.

cLSM showed proliferation of embedded MSC as evidenced by DAPI/Phalloidin stain. Vitality of embedded cells remained high over time. Articular chondrocytes and nucleus pulposus cells synthesized proteoglycans and collagen type II in the scaffolds. Also MSC embedded in the flock scaffolds differentiated and increased their chondrogenic phenotype over time.

Using cLSM and biochemical analyses we demonstrated that cells adhered and proliferated well in the new scaffolds. Furthermore we showed that the scaffolds are capable to support induction and maintenance of the chondrogenic phenotype. We conclude that flocking technology is suitable for fabrication of scaffolds for cell cultivation and cartilage tissue engineering.

We investigated the clinical, arthroscopic and biomechanical outcome of transplanting autologous chondrocytes, cultured in atelocollagen gel, for the treatment of full-thickness defects of cartilage in 28 knees (26 patients) over a minimum period of 25 months. Transplantation eliminated locking of the knee and reduced pain and swelling in all patients. The mean Lysholm score improved significantly. Arthroscopic assessment indicated that 26 knees (93%) had a good or excellent outcome. There were few adverse features, except for marked hypertrophy of the graft in three knees, partial detachment of the periosteum in three and partial ossification of the graft in one. Biomechanical tests revealed that the transplants had acquired a hardness similar to that of the surrounding cartilage. We conclude that transplanting chondrocytes in a newly-formed matrix of atelocollagen gel can promote restoration of the articular cartilage of the knee.

Metaphyseal fracture healing is important in joint-adjacent fractures and appears to differ from diaphyseal healing. We recently found that a biomaterial delivering bone morphogenic protein-2 (BMP-2) and zoledronic acid (ZA) healed the metaphyseal bone in a tibial defect but failed closing the cortical defect. In this study we added a BMP-2 soaked collagen membrane to study cortical healing from the muscle tissue surrounding the bone. We used SD rats and a 4.5 mm metaphyseal circular tibial defect. In group 1 (G1), a porous gelatin-calcium sulphate-hydroxyapatite (GCH) biomaterial containing rhBMP-2 and ZA was used to fill the defect (GCH+5 μg BMP-2+10 μg ZA). In group 2 (G2), we used a collagen membrane (2 μg BMP-2) to cover the GCH filled defect (GCH+3μg BMP+10 μg ZA). Group 3 (G3) was an empty control. Animals were sacrificed after 8-weeks and bone regeneration was evaluated with micro-CT and histology. In both G1 (P<0.001) and G2 (p<0.001) a significantly higher mineralized volume was found in the defect compared to empty G3. In G2 higher mineralized volume was found in the cortical region compared to both G1 (p<0.01) and G3 (p<0.001) as seen via micro-CT. Histologically, G1 and G2 showed islands of trabecular bone in the defect peripherally but only G2 showed cortical healing. G3 was empty in the middle but showed healed cortex. In conclusion, GCH can be used to deliver BMP-2 and ZA to promote metaphyseal bone growth. A membrane (CM) doped with low dose BMP-2 improved cortical regeneration.

Aims

Several artificial bone grafts have been developed but fail to achieve anticipated osteogenesis due to their insufficient neovascularization capacity and periosteum support. This study aimed to develop a vascularized bone-periosteum construct (VBPC) to provide better angiogenesis and osteogenesis for bone regeneration.

Objectives

Distraction osteogenesis (DO) mobilises bone regenerative potential and avoids the complications of other treatments such as bone graft. The major disadvantage of DO is the length of time required for bone consolidation. Mesenchymal stem cells (MSCs) have been used to promote bone formation with some good results.

Objectives

The present study describes a novel technique for revitalising allogenic intrasynovial tendons by combining cell-based therapy and mechanical stimulation in an ex vivo canine model.

Objectives

Effects of insulin-like growth factor 1 (IGF1), fibroblast growth factor 2 (FGF2) and bone morphogenetic protein 2 (BMP2) on the expression of genes involved in the proliferation and differentiation of osteoblasts in culture were analysed. The best sequence of growth factor addition that induces expansion of cells before their differentiation was sought.

This article reviews the current knowledge of the intervertebral disc (IVD) and its association with low back pain (LBP). The normal IVD is a largely avascular and aneural structure with a high water content, its nutrients mainly diffusing through the end plates. IVD degeneration occurs when its cells die or become dysfunctional, notably in an acidic environment. In the process of degeneration, the IVD becomes dehydrated and vascularised, and there is an ingrowth of nerves. Although not universally the case, the altered physiology of the IVD is believed to precede or be associated with many clinical symptoms or conditions including low back and/or lower limb pain, paraesthesia, spinal stenosis and disc herniation.

New treatment options have been developed in recent years. These include biological therapies and novel surgical techniques (such as total disc replacement), although many of these are still in their experimental phase. Central to developing further methods of treatment is the need for effective ways in which to assess patients and measure their outcomes. However, significant difficulties remain and it is therefore an appropriate time to be further investigating the scientific basis of and treatment of LBP.

Objectives

Matrix-assisted autologous chondrocyte transplantation (MACT) has been developed and applied in the clinical practice in the last decade to overcome most of the disadvantages of the first generation procedures. The purpose of this systematic review is to document and analyse the available literature on the results of MACT in the treatment of chondral and osteochondral lesions of the knee.

In the field of tissue engineering (TE), mainly two approaches have been widely studied and utilised throughout the last two decades. Ovsianikov et al. proposed a third strategy for tissue engineering to combine the advantages of the scaffold-based and scaffold-free approach [1]. We utilise the third strategy for TE by fabrication of cell spheroids that are reinforced by microscaffolds, called tissue units (TUs). Aim of the presented study is to differentiate TUs towards a chondrogenic phenotype to show the self-assembly of a millimetre sized cartilage-like tissue in a bottom-up TE approach in vitro. Two-Photon polymerization (2PP) was utilised to fabricate highly porous microscaffolds with a diameter of 300 µm. The biocompatible and biodegradable, resin Degrad INX (supplied from Xpect INX, Ghent, Belgium) was used for 3D-printing. Each microscaffold was seeded with 4000 human adipose derived stem cells (hASCs) in low-adhesive 96-well plates to allow spheroid formation. TUs were differentiated towards the chondrogenic lineage by application of chondrogenic media, subsequently merged in a cylindrical agarose mold, to fuse into a connected tissue with a diameter of ~1.8 mm and a height of 8 mm. The characterization of TUs differentiated towards the chondrogenic phenotype included gene expression and protein analysis. Furthermore, immunohistochemically staining for Collagen II and Alcian blue staining were performed to investigate the matrix deposition and fusion of the self-assembled tissue. Our results suggest that the utilised method could be a promising approach for a variety of tissue engineering approaches, due to the good applicability to a defect side combined with the self-assembly properties of the TUs. Furthermore, the differentiation potential of hASCs is not limited to chondrogenic lineages only, which could pave the way to further TE applications in the future. Acknowledgements:. This research work was financially supported by the European Research Council (Consolidator Grant 772464 A.O.)

The treatment of osteochondral lesions and osteoarthritis remains an ongoing clinical challenge in orthopaedics. This review examines the current research in the fields of cartilage regeneration, osteochondral defect treatment, and biological joint resurfacing, and reports on the results of clinical and pre-clinical studies. We also report on novel treatment strategies and discuss their potential promise or pitfalls. Current focus involves the use of a scaffold providing mechanical support with the addition of chondrocytes or mesenchymal stem cells (MSCs), or the use of cell homing to differentiate the organism’s own endogenous cell sources into cartilage. This method is usually performed with scaffolds that have been coated with a chemotactic agent or with structures that support the sustained release of growth factors or other chondroinductive agents. We also discuss unique methods and designs for cell homing and scaffold production, and improvements in biological joint resurfacing. There have been a number of exciting new studies and techniques developed that aim to repair or restore osteochondral lesions and to treat larger defects or the entire articular surface. The concept of a biological total joint replacement appears to have much potential.

Cite this article: Bone Joint Res 2013;2:193–9.

Objectives

There is increasing application of bone morphogenetic proteins (BMPs) owing to their role in promoting fracture healing and bone fusion. However, an optimal delivery system has yet to be identified. The aims of this study were to synthesise bioactive BMP-2, combine it with a novel α-tricalcium phosphate/poly(D,L-lactide-co-glycolide) (α-TCP/PLGA) nanocomposite and study its release from the composite.

Recently, technologies to culture one or more cell types in three dimensions have attracted a great deal of attention in tissue engineering. Particularly, the improved viability, self-renewal capacity, and differentiation potential have been reported for stem cell spheroids. However, it is crucial to modulate spheroid functions with instructive signals to use multi-cellular spheroids in tissue engineering. We have been developing ECM-mimicking fibrous materials decorated with cell-instructive cues, which were incorporated within 3D stem cell spheroids to fine-tune their functions as modular building blocks for bottom-up tissue-engineering applications. In particular, we created composite spheroids of human adipose-derived stem cells (hADSCs) incorporating nanofibers coated with instructive signal of either transforming growth factor-β3 or bone morphogenetic growth factor-2 for chondrogenesis or osteogenesis of stem cells, respectively. The bilayer structure of osteochondral tissue was subsequently mimicked by cultivating each type of spheroid inside 3D-printed construct. The in vitro chondrogenic or osteogenic differentiation of hADSCs within the biphasic construct under general media was locally regulated by each inductive component. More importantly, hADSCs from each spheroid proliferated and sprouted to form the integrated tissue with interface of bone and cartilage tissue. This approach may be applied to engineer complex tissue with hierarchically organized structure

Biofabrication is a popular technique to produce personalized constructs for tissue engineering. In this study we combined laponite (Lap), gellan gum (GG) with platelet-rich plasma (PRP) aiming to enhance the endothelial regeneration through the synergistic effects of their individual properties. Laponite has the ability to form porous three-dimensional networks mimicking the extracellular matrix structure, and PRP delivery of growth factors stimulates the endothelial cell proliferation and migration, offering a composite bioink for cell growth and support. The sustained release of these growth factors from the GG-laponite-PRP composite material over time provides a continuous source of stimulation for the cells, leading to more effective tissue engineering strategies for endothelial tissue regeneration. Four blend compositions comprising 1% w/v GG and 0.5 or 1% w/v Lap and 25% v/v PRP were combined with Wharton jelly mesenchymal stem cells (WJ-MSCs) and bioprinted into vessel-like structures with an inner diameter of 3 mm and a wall thickness of 1 mm. Stress/strain analysis revealed the elastomeric properties of the hydrogels with Young modulus values of 10 MPa. Increasing the Lap concentration led to a non-significant decrease of swelling ratio from 93 to 91%. Live/dead assay revealed cell viability of at least 76%, with the 0.5%Lap-GG viability exceeding 99% on day 21. Gradual increase of glycosaminoglycans accumulation and collagen production indicate promotion of ECM formation. The expression and membranous localization of PECAM-1 from day 7 and the granular intracellular localization of vWF after 2 weeks demonstrate in vitro endothelial functionality. In vivo subcutaneous implantation indicated the absence of any adverse immunological reactions. The results reveal the expression of both vWF and PECAM-1 by WJ-MSCs entrapped in all four construct compositions with significantly higher expression of vWF in the presence of PRP

Cells typically respond to a variety of geometrical cues in their environment, ranging from nanoscale surface topography to mesoscale surface curvature. The ability to control cellular organisation and fate by engineering the shape of the extracellular milieu offers exciting opportunities within tissue engineering. Despite great progress, however, many questions regarding geometry-driven tissue growth remain unanswered. Here, we combine mathematical surface design, high-resolution microfabrication, in vitro cell culture, and image-based characterization to study spatiotemporal cell patterning and bone tissue formation in geometrically complex environments. Using concepts from differential geometry, we rationally designed a library of complex mesostructured substrates (10. 1. -10. 3. µm). These substrates were accurately fabricated using a combination of two-photon polymerisation and replica moulding, followed by surface functionalisation. Subsequently, different cell types (preosteoblasts, fibroblasts, mesenchymal stromal cells) were cultured on the substrates for varying times and under varying osteogenic conditions. Using imaging-based methods, such as fluorescent confocal microscopy and second harmonic generation imaging, as well as quantitative image processing, we were able to study early-stage spatiotemporal cell patterning and late-stage extracellular matrix organisation. Our results demonstrate clear geometry-dependent cell patterning, with cells generally avoiding convex regions in favour of concave domains. Moreover, the formation of multicellular bridges and collective curvature-dependent cell orientation could be observed. At longer time points, we found clear and robust geometry-driven orientation of the collagenous extracellular matrix, which became apparent with second harmonic generation imaging after ∼2 weeks of culture. Our results highlight a key role for geometry as a cue to guide spatiotemporal cell and tissue organisation, which is relevant for scaffold design in tissue engineering applications. Our ongoing work aims at understanding the underlying principles of geometry-driven tissue growth, with a focus on the interactions between substrate geometry and mechanical forces

Tendon diseases are prevalent health concerns for which current therapies present limited success, in part due to the intrinsically low regenerative ability of tendons. Therefore, tissue engineering presents a potential to improve this outcome. Here, we hypothesize that a concurrent control over both biophysical and biochemical stimuli will boost the tenogenic commitment of stem cells, thus promoting regeneration. To achieve this, we combine molecularly imprinted nanoparticles (MINPs), which act as artificial amplifiers for endogenous growth factor (GF) activity, with bioinspired anisotropic hydrogels. 2. to manufacture 3D tenogenic constructs. MINPs were solid phase-imprinted using a TGF-β3 epitope as template and their affinity for the target was assessed by SPR and dot blot. Magnetically-responsive microfibers were produced by cryosectioning electrospun meshes containing iron oxide nanoparticles. The constructs were prepared by encapsulating adipose tissue-derived stem cells (ASCs), microfibers, and MINPs within gelatin hydrogels, while aligning the microfibers with an external magnetostatic field during gelation. This allows an effective modulation of hydrogel fibrillar topography, mimicking the native tissue's anisotropic architecture. Cell responses were analyzed by multiplex immunoassay, quantitative polymerase chain reaction, and immunocytochemistry. MINPs showed an affinity for the template comparable to monoclonal antibodies. Encapsulated ASCs acquired an elongated shape and predominant orientation along the alignment direction. Cellular studies revealed that combining MINPs with aligned microfibers increased TGF-β signaling via non-canonical Akt/ERK pathways and upregulated tendon-associated gene expression, contrasting with randomly oriented gels. Immunostaining of tendon-related proteins presented analogous outcomes, corroborating our hypothesis. Our results thus demonstrate that microstructural cues and biological signals synergistically direct stem cell fate commitment, suggesting that this strategy holds potential for improving tendon healing and might be adaptable for other biological tissues. The proposed concept highlights the GF-sequestering ability of MINPs which allows a cost-effective alternative to recombinant GF supplementation, potentially decreasing the translational costs of tissue engineering strategies. Acknowledgements: The authors acknowledge the funding from the European Union's Horizon 2020 under grant No. 772817; from FCT/MCTES for scholarships PD/BD/143039/2018 & COVID/BD/153025/2022 (S.P.B.T.), and PD/BD/129403/2017 (S.M.B.), co-financed by POCH and NORTE 2020, under the Portugal 2020 partnership agreement through the European Social Fund, for contract 2020.03410.CEECIND (R.M.A.D.) and project 2022.05526.PTDC; and from Xunta de Galicia for grant ED481B2019/025 (A.P.)

In the last decades, significant effort has been attempted to salvage the meniscus following injury. Basic science approaches to meniscus repair include procedures for both meniscus regeneration and meniscus healing. Regeneration of meniscal tissue focuses on filling a defect with reparative tissue, which resembles the native structure and function of the meniscus. Procedures for meniscus healing, on the other hand, aim to accomplish adhesion between the margins of a meniscal lesion, with no attempt to regenerate or replace meniscal tissue. Regeneration studies of tissue to fill a defect in the meniscus have shown interesting results, but complete restoration of the native meniscus has not yet been accomplished. Healing of a meniscal lesion has been investigated in different models although none has demonstrated reproducible healing. Therefore, different paths of investigation must be undertaken, and one of these may be the cell-therapy / tissue engineering approach. In a study from our group, we showed the capacity of chondrocyte-seeded cartilaginous scaffold to repair a bucket-handle lesion of the knee meniscus orthotopically in a large animal study. Following studies were done in order to test the potential of other scaffolds and different cell sources for the repair of the meniscal tissue. We have also evaluated the role of hypoxia in meniscal development in vitro as basis for future research in this field, as hypoxia could be be considered as a promoter for meniscal cells maturation, and opens considerably opportunities in the field of meniscus tissue engineering

Tendon is a bradytrophic and hypovascular tissue, hence, healing remains a major challenge. The molecular key events involved in successful repair have to be unravelled to develop novel strategies that reduce the risk of unfavourable outcomes such as non-healing, adhesion formation, and scarring. This review will consider the diverse pathophysiological features of tendon-derived cells that lead to failed healing, including misrouted differentiation (e.g. de- or transdifferentiation) and premature cell senescence, as well as the loss of functional progenitors. Many of these features can be attributed to disturbed cell-extracellular matrix (ECM) or unbalanced soluble mediators involving not only resident tendon cells, but also the cross-talk with immigrating immune cell populations. Unrestrained post-traumatic inflammation could hinder successful healing. Pro-angiogenic mediators trigger hypervascularization and lead to persistence of an immature repair tissue, which does not provide sufficient mechano-competence. Tendon repair tissue needs to achieve an ECM composition, structure, strength, and stiffness that resembles the undamaged highly hierarchically ordered tendon ECM. Adequate mechano-sensation and -transduction by tendon cells orchestrate ECM synthesis, stabilization by cross-linking, and remodelling as a prerequisite for the adaptation to the increased mechanical challenges during healing. Lastly, this review will discuss, from the cell biological point of view, possible optimization strategies for augmenting Achilles tendon (AT) healing outcomes, including adapted mechanostimulation and novel approaches by restraining neoangiogenesis, modifying stem cell niche parameters, tissue engineering, the modulation of the inflammatory cells, and the application of stimulatory factors. Cite this article: Bone Joint Res 2022;11(8):561–574

Aims. Minimally manipulated cells, such as autologous bone marrow concentrates (BMC), have been investigated in orthopaedics as both a primary therapeutic and augmentation to existing restoration procedures. However, the efficacy of BMC in combination with tissue engineering is still unclear. In this study, we aimed to determine whether the addition of BMC to an osteochondral scaffold is safe and can improve the repair of large osteochondral defects when compared to the scaffold alone. Methods. The ovine femoral condyle model was used. Bone marrow was aspirated, concentrated, and used intraoperatively with a collagen/hydroxyapatite scaffold to fill the osteochondral defects (n = 6). Tissue regeneration was then assessed versus the scaffold-only group (n = 6). Histological staining of cartilage with alcian blue and safranin-O, changes in chondrogenic gene expression, microCT, peripheral quantitative CT (pQCT), and force-plate gait analyses were performed. Lymph nodes and blood were analyzed for safety. Results. The results six months postoperatively showed that there were no significant differences in bone regrowth and mineral density between BMC-treated animals and controls. A significant upregulation of messenger RNA (mRNA) for types I and II collagens in the BMC group was observed, but there were no differences in the formation of hyaline-like cartilage between the groups. A trend towards reduced sulphated glycosaminoglycans (sGAG) breakdown was detected in the BMC group but this was not statistically significant. Functional weightbearing was not affected by the inclusion of BMC. Conclusion. Our results indicated that the addition of BMC to scaffold is safe and has some potentially beneficial effects on osteochondral-tissue regeneration, but not on the functional endpoint of orthopaedic interest. Cite this article: Bone Joint Res 2021;10(10):677–689

Our musculoskeletal system has a limited capacity for repair. This has led to increased interest in the development of tissue engineering and biofabrication strategies for the regeneration of musculoskeletal tissues such as bone, ligament, tendon, meniscus and articular cartilage. This talk will demonstrate how different musculoskeletal tissues, specifically cartilage, bone and osteochondral defects, can be repaired using emerging biofabrication and 3D bioprinting strategies. This will include examples from our lab where cells and/or growth factors are bioprinted into constructs that can be implanted directly into the body, to approaches where biomimetic tissues are first engineered in vitro before in vivo implantation. The efficacy of these different biofabrication strategies in different preclinical studies will be reviewed, and lessons from the relative successes and failures of these approaches to tissue regeneration will be discussed

Fractures and related complications are a common challenge in the field of skeletal tissue engineering. Vitamin D and calcium are the only broadly available medications for fracture healing, while zinc has been recognized as a nutritional supplement for healthy bones. Here, we aimed to use polaprezinc, an anti-ulcer drug and a chelate form of zinc and L-carnosine, as a supplement for fracture healing. Polaprezinc induced upregulation of osteogenesis-related genes and enhanced the osteogenic potential of human bone marrow-derived mesenchymal stem cells and osteoclast differentiation potential of mouse bone marrow-derived monocytes. In mouse experimental models with bone fractures, oral administration of polaprezinc accelerated fracture healing and maintained a high number of both osteoblasts and osteoclasts in the fracture areas. Collectively, polaprezinc promotes the fracture healing process efficiently by enhancing the activity of both osteoblasts and osteoclasts. Therefore, we suggest that drug repositioning of polaprezinc would be helpful for patients with fractures

Stem cell therapy for the intervertebral disc (IVD) is highly debated but holds great promises. From previous studies, it is known that notochordal cells are highly regenerative and may stimulate other differentiated cells to produce more matrix. Lately, a particular tissue-specific progenitor cell population has been identified in the centre of the intervertebral disc (IVD. The current hope is that these nucleus pulposus progenitor cells (NPPC) could play a particular role in IVD regeneration. Current evidence confirms the presence of these cells in murine, canine, bovine and in the human fetal/surgical samples. Noteworthy, one of the main markers to identify these cells, i.e., Tie2, is a typical marker for endothelial cells. Thus, it is not very clear what their origin and their role might be in the context of developmental biology. In human surgical specimens, their presence is, even more, obscured depending on the donor's age and the condition of the IVD and other yet unknown factors. Here, I revisit the recent literature on regenerative cells identified for the IVD in the past decades. Current evidence how these NPPC can be isolated and detected in various species and tissues will be recapitulated. Future directions will be provided on how these progenitor cells could be used for regenerative medicine and tissue engineering

The use of mesenchymal stromal cells (MSCs) in regenerative medicine and tissue engineering is well established, given their properties of self-renewal and differentiation. However, several studies have shown that these properties diminish with age, and understanding the pathways involved are important to provide regenerative therapies in an ageing population. In this PRISMA systematic review, we investigated the effects of chronological donor ageing on the senescence of MSCs. We identified 3023 studies after searching four databases including PubMed, Web of Science, Cochrane, and Medline. Nine studies met the inclusion and exclusion criteria and were included in the final analyses. These studies showed an increase in the expression of p21, p53, p16, ROS, and NF- B with chronological age. This implies an activated DNA damage response (DDR), as well as increased levels of stress and inflammation in the MSCs of older donors. Additionally, highlighting the effects of an activated DDR in cells from older donors, a decrease in the expression of proliferative markers including Ki67, MAPK pathway elements, and Wnt/ -catenin pathway elements was observed. Furthermore, we found an increase in the levels of SA- -galactosidase, a specific marker of cellular senescence. Together, these findings support an association between chronological age and MSC senescence. The precise threshold for chronological age where the reported changes become significant is yet to be defined and should form the basis for further scientific investigations. The outcomes of this review should direct further investigations into reversing the biological effects of chronological age on the MSC senescence phenotype

Symptomatic articular cartilage defects are one of the most common knee injuries, arising from acute trauma, overuse, ligamentous instability, malalignment, meniscectomy, osteochondritis dissecans. Surgical treatment options include bone marrow–stimulating techniques such as abrasion arthroplasty and microfracture, osteochondral mosaicplasty, corrective osteotomy, cartilage resurfacing techniques and tissue engineering techniques using combinations of autologous cells (chondrocytes and mesenchymal stem cells), bioscaffolds, and growth factors. Matrix induced autologous chondrocyte implantation (MACI) is considered the most surgically simple form of autologous chondrocyte implantation. Our group has involved in the development of MACI since 2000 and has led to the FDA approval of MACI as the first tissue engineering product for cartilage repair in 2016. In this article, we have documented the characterisation of autologous chondrocytes, the surgical procedure of MACI and the long term clinical assessment (15 years) of patients with treatment of MACI. We have also reported the retrospective survey in patients with MACI in Australia. Our results suggest that MACI has gained good to excellent long term clinical outcome and probably can delay total knee replacement. However, restoration of hyaline-like cartilage by MACI may be interrupted by the osteoarthritic condition of the joint in patients with progressed osteoarthritis. In addition, because articular cartilage and subchondral bone are considered a single functional unit that is essential for joint function, many cartilage repair technologies including MACI and microfractures have failed short to address the functional barrier structure of osteochondral unit. Further studies are required to develop tissue engineering osteochondral construct that is able to fulfil the function of articular cartilage-subchondral bone units

The application of immune regenerative strategies to deal with unsolved pathologies, such as tendinopathies, is getting attention in the field of tissue engineering exploiting the innate immunomodulatory potential of stem cells [1]. In this context, Amniotic Epithelial Cells (AECs) represent an innovative immune regenerative strategy due to their teno-inductive and immunomodulatory properties [2], and because of their high paracrine activity, become a potential stem cell source for a cell-free treatment to overcome the limitations of traditional cell-based therapies. Nevertheless, these immunomodulatory mechanisms on AECs are still not fully known to date. In these studies, we explored standardized protocols [3] to better comprehend the different phenotypic behavior between epithelial AECs (eAECs) and mesenchymal AECs (mAECs), and to further produce an enhanced immunomodulatory AECs-derived secretome by exposing cells to different stimuli. Hence, in order to fulfill these aims, eAECs and mAECs at third passage were silenced for CIITA and Nrf2, respectively, to understand the role of these molecules in an inflammatory response. Furthermore, AECs at first passage were seeded under normal or GO-coated coverslips to study the effect of GO on AECs, and further exposed to LPS and/or IL17 priming to increase the anti-inflammatory paracrine activity. The obtained results demonstrated how CIITA and Nrf2 control the immune response of eAECs and mAECs, respectively, under standard or immune-activated conditions (LPS priming). Additionally, GO exposition led to a faster activation of the Epithelial-Mesenchymal transition (EMT) through the TGFβ/SMAD signaling pathway with a change in the anti-inflammatory properties. Finally, the combinatory inflammatory stimuli of LPS+IL17 enhanced the paracrine activity and immunomodulatory properties of AECs. Therefore, AECs-derived secretome has emerged as a potential treatment option for inflammatory disorders such as tendinopathies. Acknowledgement: This research is part of the P4FIT project ESR1, funded under the H2020-ITN-EJD-Marie-Skłodowska-Curie grant agreement 955685

Traumatic acute or chronic tendon injuries are a wide clinical problem in modern society, resulting in important economic burden to the health system and poor quality of life in patients. Due to the low cellularity and vascularity of tendon tissue the repair process is slow and inefficient, resulting in mechanically, structurally, and functionally inferior tissue. Tissue engineering and regenerative medicine are promising alternatives to the natural healing process for tendon repair, especially in the reconstruction of large damaged tissues. The aim of TRITONE project is to develop a smart, bioactive implantable 3D printed scaffold, able to reproduce the structural and functional properties of human tendon, using FDA approved materials and starting from MSC and their precursor, MPC cell mixtures from human donors. Total cohort selected in the last 12 months was divided in group 1 (N=20) of subjects with tendon injury and group 2 (N=20) of healthy subject. Groups were profiled and age and gender matched. Inclusion criteria were age>18 years and presence of informed consent. Ongoing pregnancy, antihypertensive treatment, cardiovascular diseases, ongoing treatment with anti-aggregants, acetylsalicylic-acid or lithium and age<18 years were exclusion criteria. Firstly, we defined clinical, biological, nutritional life style and genetic profile of the cohort. The deficiency of certain nutrients and sex hormonal differences were correlated with tendon-injured patients. It was established the optimal amount of MPC/MSC human cell (collected from different patients during femoral neck osteotomy). Finally, most suitable biomaterials for tendon regeneration and polymer tendon-like structure were identified. Hyaluronic acid, chemical surface and soft-molecular imprinting (SOFT-MI) was used to functionalize the scaffold. These preliminary results are promising. It will be necessary to enroll many more patients to identify genetic status connected with the onset of tendinopathy. The functional and structural characterization of smart bioactive tendon in dynamic environment will represent the next project step

Periosteal mesenchymal stem cells (PMSC) are an emerging niche of stem cells to enhance bone healing by tissue engineering process. They have to be differentiated into osteoprogenitors in order to synthesize new bone matrix. In vitro differentiation with specific differentiation medium (DM) is not exactly representative of what occurs in vivo. The interaction between PMSC and growth factors (GF) present in biological matrix is somewhat less understood. The goal of this study is to explore the possibility of spontaneous PMSC differentiation in contact with different biological matrices without DM. 500.000 porcine PMSC were seeded on 6-well plates and cultured with proliferation medium (PM). When reaching 80% confluence, biological samples (n=3) of demineralized bone matrix (DBM), decellularized porcine bone allograft (AOp), human bone allograft (AOh), human periosteum (HP) and human fascia lata (HFL) were added. Negative and positive control wells included cells with only PM or DM, respectively. The differentiation progress was assessed by Alizarin Red staining at days 7, 14 and 21. Bone morphogenetic protein content (BMP 2, 4, 5, 6, 7, 8, 9 and 11) of each sample was also investigated by western blot. Alizarin red highlighted bone nodules neoformation on wells containing AOp, AOh and DBM, like positive controls. HP and HFL wells did not show any nodules. These results are correlated to a global higher BMP expression profile in AOp than in HP and HFL but not statistically significant (p=0.38 and p>.99, respectively). The highest expression in each tissue was that of BMP2 and BMP7, which play an important role in osteoinduction. PMSC are well known to participate to bone formation but, despite BMP presence in HP and HFL, they did not permit to achieve osteogenesis alone. The bone contact seems to be essential to induce in vitro differentiation into osteoprogenitors

Degenerative disc disease, associated to low back pain, afflicts more than 50% of humans, and represents a major healthcare problem, especially for the pathology initiation. Current treatments range from conservative strategies to more invasive surgical techniques, such as disc removal and vertebral fusion. In the Intervertebral Disease (IVD) the nucleus pulposus (NP) degeneration is a key factor for the pathology initiation. Several tissue engineering approaches aiming to restore the appropriate NP cell (NPCs) and matrix content, were attempted by using adult stromal cells either from bone marrow or adipose tissue, chondrocytes, notochordal cells and more recently also pluripotent stem cells. However, none was fully satisfactory since the NP acid and a-vascularized environment appeared averse to the implanted heterologous cells. Several studies demonstrated the efficacy of platelet derivatives such as platelet rich plasma (PRP) in promoting the regeneration of connective tissues. We investigated the efficacy of PRP on NPCs proliferation and differentiation with the goal to propose the direct stimulation of resident cells (stimulation of endogenous cells – less invasive surgical procedure) or the implantation of NPCs expanded in vitro in the presence of PRP as therapeutic agents in IVD degeneration. NPCs were isolated from small fragments of NP explants, cultivated in medium supplemented with PRP or FCS (standard condition control) and characterized by FACS analysis for the expression of the typical mesenchymal stem cells markers CD34, CD44, CD45, CD73, CD90 and CD105. NPCs cultured in PL showed a phenotypic profile like the cells cultured in FCS. However, compared to NPCs expanded in the presence of FCS, NPCs expanded in PRP showed a much better proliferation and differentiation capacity. NPCs differentiation was evaluated by the cell ability to produce an organized metachromatic cartilaginous matrix, confirmed by the positive immunohistochemical staining for chondrogenic markers

Although autografts represent the gold standard for anterior cruciate ligament (ACL) reconstruction, tissue-engineered ACLs provide a prospect to minimize donor site morbidity and limited graft availability. This given study characterizes the ligamentogenesis in embroidered poly(L-lactide-co-ε-caprolactone) (P(LA-CL)) / polylactic acid (PLA) constructs using a dynamic nude mice xenograft model. (P(LA-CL))/PLA scaffolds remained either untreated (co) or were functionalized by gas fluorination (F), collagen foam cross-linked with hexamethylene diisocyanate (HMDI) (coll), or gas fluorination combined with the foam (F+coll). Cell free constructs or those seeded for 1 week with lapine ACL ligamentocytes were implanted into nude mice for 12 weeks. Following explantation, biomechanical properties, cell vitality and content, histopathology of scaffolds (including organs: liver, kidney, spleen), sulphated glycosaminoglycan (sGAG) contents and biomechanical properties were assessed. Implantation of the scaffolds did not negatively affect mice weight development and organs, indicating biocompatibility. All scaffolds maintained their size and shape for the duration of the implantation. A high cell viability was detected in the scaffolds prior to and following implantation. Coll or F+coll scaffolds seeded with cells yielded superior macroscopic properties when compared to the controls. Mild signs of inflammation (foreign-body giant cells, hyperemia) were limited to scaffolds without collagen. Microscopical score values and sGAG content did not differ significantly. Although remaining stable in vivo, elastic modulus, maximum force, tensile strength and strain at Fmax were significantly lower in the in vivo compared to the samples cultured 1 week in vitro, but did not differ between scaffold subtypes, except for a higher maximum force in F+coll compared with F samples (in vivo). Scaffold functionalization with fluorinated collagen foam provides a promising approach for ACL tissue engineering. (shared first authorship). Acknowledgement: The study was supported by DFG grants SCHU1979/9-1 and SCHU1979/14-1

The reliable production of _in vitro_ chondrocytes that faithfully recapitulate _in vivo_ development would be of great benefit for orthopaedic disease modelling and regenerative therapy(1,2). Current efforts are limited by off-target differentiation, resulting in a heterogeneous product, and by the lack of comparison to human tissue, which precludes detailed evaluation of _in vitro_ cells(3,4). We performed single-cell RNA-sequencing of long bones dissected from first-trimester fetal limbs to form a detailed ‘atlas’ of endochondral ossification. Through 100-gene in-situ sequencing, we placed each sequenced cell type into its anatomical context to spatially resolve the process of endochondral ossification. We then used this atlas to perform deconvolution on a series of previously published bulk transcriptomes generated from _in vitro_ chondrogenesis protocols to evaluate their ability to accurately produce chondrocytes. We then applied single-nuclear RNA-sequencing to cells from the best performing protocol collected at multiple time points to allow direct comparison between the differentiation of _in vitro_ and _in vivo_ cells. We captured 275,000 single fetal cells, profiling the development of chondrocytes from multipotent mesenchymal progenitors to hypertrophic cells at full transcriptomic breadth. Using this atlas as the ground truth for evaluating _in vitro_ cells, we found substantial variability in cell states produced by each protocol, with many showing little similarity to _in vivo_ cells, and all exhibiting off-target differentiation. Trajectory alignment between _in vivo_ and _in vitro_ single-cell data revealed key differences in gene expression dynamics between _in vitro_ and _in vivo cells,_ with several osteoblastic transcription factors erroneously unregulated _in vitro,_ including _FOXO1._. Using this information, we inhibited _FOXO1_ in culture to successfully increase chondrocyte yield _in vitro._. This study presents a new framework for evaluating tissue engineering protocols, using single-cell data to drive improvement and bring the prospect of true engineered cartilage closer to reality

Regeneration of bone defects in elderly patients is limited due to the decreased function of bone forming cells and compromised tissue physiology. Previous studies suggested that the regenerative activity of stem cells from aged tissues can be enhanced by exposure to young systemic and tissue microenvironments. The aim of our project was to investigate whether extracellular matrix (ECM) engineered from human induced pluripotent stem cells (hiPSCs) can enhance the bone regeneration potential of aged human bone marrow stromal cells (hBMSCs). ECM was engineered from hiPSC-derived mesenchymal-like progenitors (hiPSC-MPs), as well as young (<30 years) and aged (>70 years) hBMSCs. ECM structure and composition were characterized before and after decellularization using immunofluorescence and biochemical assays. Three hBMSCs of different ages were cultured on engineered ECMs. Growth and differentiation responses were compared to tissue culture plastic, as well as to collagen and fibronectin coated plates. Decellularized ECMs contained collagens type I and IV, fibronectin, laminin and < 5% residual DNA, suggesting efficient cell elimination. Cultivation of young and aged hBMSCs on the hiPSC-ECM in osteogenic medium significantly increased hBMSC growth and markers of osteogenesis, including collagen deposition, alkaline phosphatase activity, bone sialoprotein expression and matrix mineralization compared to plastic controls and single protein substrates. In aged BMSCs, matrix mineralization was only detected in ECM cultures in osteogenic medium. Comparison of ECMs engineered from hiPSC-MPs and hBMSCs of different ages suggested similar structure, composition and potential to enhance osteogenic responses in aged BMSCs. Engineered ECM induced a higher osteogenic response compared to specific matrix components. Our studies suggest that aged BMSCs osteogenic activity can be enhanced by culture on engineered ECM. hiPSCs represent a scalable cell source, and tissue engineering strategies employing engineered ECM materials could potentially enhance bone regeneration in elderly patients

In this work, we combined tissue engineering and gene therapy technologies to develop a therapeutic platform for bone regeneration. We have developed photothermal fibrin-based hydrogels that incorporate degradable CuS nanoparticles (CuSNP) which transduce incident near-infrared (NIR) light into heat. A heat-activated and rapamycin-dependent transgene expression system was incorporated into mesenchymal stem cells to conditionally control the production of bone morphogenetic protein 2 (BMP-2). Genetically engineered cells were entrapped in the photothermal hydrogels. In the presence of rapamycin, photoinduced mild hyperthermia induced the release of BMP-2 from the NIR responsive cell constructs. Transcriptome analysis of BMP-2 expressing cells showed a signature of induced genes related to stem cell proliferation and angiogenesis. We next generated 4 mm diameter calvarial defects in the left parietal bone of immunocompetent mice. The defects were filled with NIR-responsive hydrogels entrapping cells that expressed BMP-2 under the control of the gene circuit. After one and eight days, rapamycin was administered intraperitoneally followed by irradiation with an NIR laser. Ten weeks after implantation, the animals were euthanized and samples from the bone defect zone were processed for histological analysis using Masson's trichrome staining and for immunohistochemistry analyses using specific CD31 and CD105 antibodies. Samples from mice that were only administered rapamycin or vehicle or that were only NIR-irradiated showed the persistence of fibrous tissue bridging the defect. In animals that were treated with rapamycin, NIR irradiation of implants resulted in the formation of new mineralized tissue with a high degree of vascularization, thus indicating the therapeutic potential of the approach. Acknowledgements: This research was supported by grants RTI2018-095159-B-I00 and PID2021-126325OB-I00 (MCIN/AEI/10.13039/501100011033 and “ERDF A way of making Europe”), by grant P2022/BMD- 7406 (Regional Government of Madrid). M.A.L-J. is the recipient of predoctoral fellowship PRE2019-090430 (MCIN/AEI/10.13039/501100011033)

Worldwide, tendon disorders are one of the main causes of disability that decrease the quality of life of individuals and represent a substantial economic burden on society. Currently, the main therapies used for tendon injuries are not able to restore tendon functionality, and due to tendons' hypovascular and hypocellular nature, they present a reduced healing capacity, which also limits the success of the available therapies. In order to discover new therapies, extracellular vesicles (EVs), key players in cell-cell communication, have been widely explored for tissue engineering and regenerative medicine applications. Thus, the aim of this study is to assess the role of EVs derived from platelets in stem cell tenogenic commitment using a bioengineered tendon in vitro model for potential use as tendon therapeutic agents. Biomimetic platelet-derived EVs were produced by freeze-thaw cycles of platelets and isolation at different centrifugation speed. To recreate the architecture of tendons, a 3D system consisting of electrospun anisotropic nanofiber scaffolds coated with collagen encapsulating human adipose stem cells (hASCs) and different types of platelet-derived EVs, were produced. Then, the influence of the tendon-mimetic constructs and the distinct EVs populations in the hASCs tenogenic differentiation were assessed over culture time. We observed that the hASCs on the nanofibrous tendon scaffolds, show high cytoskeleton anisotropic organization that is characteristic of tenocytes. Moreover, acting as biological cues, platelet-derived EVs boosted hASCs tenogenic commitment, supported by the increased gene expression of tendon-related markers (SCX and TNMD). Additionally, EVs enhanced the deposition of tendon like extracellular matrix (ECM), as evidenced by the increased gene expression of ECM-related markers such as COL1, COL3, DCN, TNC, and MMP-3, which are fundamental for ECM synthesis and degradation balance. Moreover, EVs induced lower collagen matrix contraction on hASCs, which has been related with lower myofibroblast differentiation. Overall, the results revealed that EVs are capable of modulating stem cells' behavior boosting their tenogenic commitment, through the increased expression of healthy tendon cell markers, potentiating ECM deposition and decreasing cell contractility. Therefore, platelet EVs are a promising biochemical tool, worthy to be further explored, as paracrine signaling that might potentiate tendon repair and regeneration