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

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

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.

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.

The ability of mesenchymal stem cells (MSCs) to differentiate in vitro into chondrocytes, osteocytes and myocytes holds great promise for tissue engineering. Skeletal defects are emerging as key targets for treatment using MSCs due to the high responsiveness of bone to interventions in animal models. Interest in MSCs has further expanded in recognition of their ability to release growth factors and to adjust immune responses. Despite their increasing application in clinical trials, the origin and role of MSCs in the development, repair and regeneration of organs have remained unclear. Until recently, MSCs could only be isolated in a process that requires culture in a laboratory; these cells were being used for tissue engineering without understanding their native location and function. MSCs isolated in this indirect way have been used in clinical trials and remain the reference standard cellular substrate for musculoskeletal engineering. The therapeutic use of autologous MSCs is currently limited by the need for ex vivo expansion and by heterogeneity within MSC preparations. The recent discovery that the walls of blood vessels harbour native precursors of MSCs has led to their prospective identification and isolation. MSCs may therefore now be purified from dispensable tissues such as lipo-aspirate and returned for clinical use in sufficient quantity, negating the requirement for ex vivo expansion and a second surgical procedure. In this annotation we provide an update on the recent developments in the understanding of the identity of MSCs within tissues and outline how this may affect their use in orthopaedic surgery in the future. Cite this article: Bone Joint J 2014;96-B:291–8

Anterior cruciate ligament (ACL) reconstruction is the current standard of care for ACL tears. However, the results are not consistently successful, autografts or allografts have certain disadvantages, and synthetic grafts have had poor clinical results. The aim of this study was to determine the efficacy of tissue engineering decellularized tibialis tendons by recellularization and culture in a dynamic tissue bioreactor. To determine if recellularization of decellularized tendons combined with mechanical stimulation in a bioreactor could replicate the mechanical properties of the native ACL and be successfully used for ACL reconstruction in vivo. Porcine tibialis tendons were decellularized and then recellularized with human adult bone marrow-derived stem cells. Tendons were cultured in a tissue bioreactor that provided biaxial cyclic loading for up to 7 days. To reproduce mechanical stresses similar to hose experienced by the ACL within the knee joint, the tendons were subjected to simultaneous tension and torsion in the bioreactor. Expression of tendon-specific genes, and newly synthesized collagen and glycosaminoglycan (GAG) were used to quantify the efficacy of recellularization and dynamic bioreactor culture. The mechanical strength of recellularized constructs was measured after dynamic stimulation. Finally, the tissue-engineered tendons were used to reconstruct the ACL in mini-pigs and mechanical strength was assessed after three months. Dynamic bioreactor culture significantly increased the expression of tendon-specific genes, the quantity of newly synthesized collagen and GAG, and the tensile strength of recellularized tendons. After in vivo reconstruction, the tensile strength of the tissue-engineered tendons increased significantly up to 3 months after surgery and were within 80% of the native strength of the ACL. Our translational study indicates that the recellularization and dynamic mechanical stimuli can significantly enhance matrix synthesis and mechanical strength of decellularized porcine tibialis tendons. This approach to tissue engineering can be very useful for ACL reconstruction and may overcome some of the disadvantages of autografts and allografts

Millions of medical devices made of synthetic or modified natural materials all trigger a similar reaction—the foreign body reaction. Biocompatibility, for materials that pass routine cytoxicity assays, is largely associated with a mild foreign body reaction. I.e. a thin, avacular, collagenous, non-adherent foreign body capsule. The implant is incorporated into a dead-zone of acellular scar. The contemporary tissue engineering paradigm would suggest that synthetic polymers and scaffolds lacking cellular, biomolecule or biomimetic elements will give this same fibrotic, avascular healing reaction. In this talk, a synthetic biomaterial will be described that readily integrates into tissue and may stimulate spontaneous reconstruction of tissue. The material is fabricated by a process called sphere-templating and it can be made from many synthetic polymers including hydrogels, silicones and polyurethanes. All pores are identical in size and interconnected. Studies from our group have shown optimal healing (as suggested by extensive vascularity and minimal fibrosis) for spherical pores of 30–40 m size. The integrative healing noted is independent of biomaterial. Similar results are observed with sphere-templated silicone rubber and pHEMA hydrogel. In addition, surface chemical modification of the hydrogel with carbonyl diimidazole, or immobilisation on the hydrogel of collagen I or laminin did not change the healing response. Also, good healing results have been seen upon implantation in skin (subcutaneous, percutaneous), heart muscle, sclera, skeletal muscle, bone and vaginal wall. We consistently find the pore spaces heavily populated by monocytic cells that stain for macrophage cell surface markers. However, at long implantation times (16 or more weeks), the ability to stain for macrophage surface markers decreases. It could be possible that these cells populating the implants are differentiating into other tissues. Thus, such materials may represent a path to cell-free tissue engineering. Others have seen similar healing results, via completely different materials strategies, generally involving biological molecules. The in vivo results from our group and related results from other groups suggest we are on the cusp of a revolution in healing, biomaterials integration and tissue reconstruction. Also, the boundaries between biomaterials and tissue engineering continue to blur

Tissue engineering by self-assembly is a technique that consists of growing cells on surfaces made of thermoresponsive polymers, that allow the production of contiguous cell sheets by simply lowering the temperature below the polymer's low critical solution temperature. In this approach cell-cell junctions and deposited extracellular matrix (ECM) remain intact, which provides a better cell localisation at the site of injury. However, these systems lack the possibility to fabricate multi-layered and three-dimensional cell sheets that would better recapitulate native tissues. Moreover, the fabrication of ECM-rich cell sheets would be highly desirable. This limitation could be overcome by inducing macromolecular crowding (MMC) conditions. Herein we venture to fabricate electrospun thermoresponsive nanofibres to sustain the growth and detachment of ECM-rich tissue substitutes in the presence of a MMC microenvironment. A copolymer of 85% poly-N-isopropylacrylamide and 15% N-tert-butylacrylamide (pNIPAAm/NTBA) were used for all experiments. To create aligned nanofibers, the polymer was electrospun and collected on a mandrel rotating at 2000 rpm. Human adipose derived stem cells (hADSC) were treated with media containing macromolecular crowders to enhance matrix deposition. Cell viability and morphology were assessed, and immunocytochemistry was conducted in order to estimate matrix deposition and composition. Adipogenic, osteogenic and chondrogenic assays were performed both with and without the presence of MMC. Non-invasive cell detachment was enabled by decreasing the temperature of culture to 10 °C for 20 minutes. The electrospinning process resulted in the production of pNIPAm/NTBA fibres in the diameter range from 1 to 2 µm and an overall alignment of 80%. Cell viability, proliferation and metabolic activity revealed that hADSCs were able to grow on the thermoresponsive scaffold. The cells were able to detach as an intact cell sheet in presence of MMC. Moreover, it was demonstated that MMC, by a volume extrusion effect, enhances Collagen type I deposition, which is one of the main components of the ECM. Histological analysis revealed that in the presence of MMC the cells were able to self-assembled into three dimensional multi-layers. The cells were able to differentiate towards the osteogenic and adipogenic lineage in the presence of MMC. Interestingly we were able to fabricate three-dimensional chondrogenic cell sheet both with and without MMC. Collectively the pNIPAm/NTBA thermoresponsive fibres were able to sustain the growth and the detachment of ECM-rich multi-layered cell sheets. The pNIPAm/NTBA fibres were able to successfully sustain growth and detachment of ECM-rich tissue equivalents. We believe that replacement, repair and restoration of tissue function can be accomplished best using cells that create their own tissue-specific extracellular matrix with a precision and stoichiometric efficiency still unmatched by man-made devices

Recently, the osteoregenerative properties of allograft have been enhanced by addition of autogenous skeletal stem cells to treat orthopaedic conditions characterised by lost bone stock. There are multiple disadvantages to allograft, and trabecular tantalum represents a potential alternative. This metal is widely used, although in applications where there is poor initial stability, or when it is used in conjunction with bone grafting, loading may need to be limited until sound integration has occurred. Strategies to speed up implant incorporation to surrounding bone are therefore required. This may improve patient outcomes, extending the clinical applications of tantalum as a substitute for allograft. Aim. To use tissue engineering strategies to enhance the reconstructive properties of tantalum, as an alternative to allograft. Methods. Human bone marrow stromal cells (5×10. 5. cells/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 detail cell adherence, proliferation and phenotype. Results. Cells displayed extensive adherence and proliferation throughout trabecular tantalum. Samples cultured in osteogenic conditions showed abundant matrix production. Electron microscopy confirmed significant cellular growth through tantalum to a depth of 5mm. In contrast to cells cultured with allograft in both basal and osteogenic conditions, cell proliferation and biochemical assays showed significantly higher activity with tantalum than allograft. Furthermore, 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. Conclusions. These studies demonstrate trabecular tantalum supports cell growth and osteogenic differentiation at least as well as allograft. Trabecular tantalum represents a good alternative to allograft for tissue engineering osteoregenerative strategies in the context of lost bone stock. Further mechanical testing and in vivo studies are on-going

Cellular therapies play an important role in tendon tissue engineering with tenocytes being described as the most prominent cell population if available in large numbers. However, in vitro expansion of tenocytes in standard culture leads to phenotypic drift and cellular senescence. Recent work suggests that maintenance of tenogenic phenotype in vitro can be achieved by recapitulating different aspects of the native tendon microenvironment. One approach used to modulate the in vitro microenvironment and enhance extracellular matrix (ECM) deposition is macromolecular crowding (MMC). MMC is based on the addition of inert macromolecules to the culture media mimicking the dense extracellular matrix. In addition, as tendon has been described to be a relatively avascular and hypoxic tissue and low oxygen tension can stimulate collagen synthesis and cross-linking, we venture to assess the synergistic effect of MMC and low oxygen tension on human tenocyte phenotype maintenance by enhancing synthesis and deposition of tissue-specific ECM. Human tendons were kindly provided from University Hospital Galway, after obtaining appropriate licenses, ethical approvals and patient consent. Afterwards, tenocytes were extracted using the migration method. Experiments were conducted at passage three. Optimization of MMC conditions was assessed using 50 to 500 μg/ml carrageenan (Sigma Aldrich, UK). For variable oxygen tension cultures, tenocytes were incubated in a Coy Lab (USA) hypoxia chamber. ECM synthesis and deposition were assessed using SDS-PAGE (BioRad, UK) and immunocytochemistry (ABCAM, UK) analysis. Protein analysis for Scleraxis (ABCAM, UK) was performed using western blot. Gene analysis was conducted using a gene array (Roche, Ireland). Cell morphology was assessed using bright-field microscopy. All experiments were performed at least in triplicate. MINITAB (version 16, Minitab, Inc.) was used for statistical analysis. Two-sample t-test for pairwise comparisons and ANOVA for multiple comparisons were conducted. SDS-PAGE and immunocytochemistry analysis demonstrated that human tenocytes treated with the optimal MMC concentration at 2% oxygen tension showed increased synthesis and deposition of collagen type I, the major component of tendon ECM. Moreover, immunocytochemistry for the tendon-specific ECM proteins collagen type III, V, VI and fibronectin illustrated enhanced deposition when cells were treated with MMC at 2% oxygen tension. In addition, protein analysis revealed elevated dexpression of the tendon-specific protein Sclearaxis, while a detailed gene analysis revealed upregulation of tendon-related genes and downregulation of trans-differentiation markers again when cells cultured with MMC at 2% oxygen tension. Finally, low oxygen tension and MMC did not affect the metabolic activity, proliferation and viability of human tenocytes. Collectively, results suggest that the synergistic effect of MMC and low oxygen tension can accelerate the formation of ECM-rich substitutes, which stimulates tenogenic phenotype maintenance. Currently, the addition of substrate aligned topography together with MMC and hypoxia is being investigated in this multifactorial study for the development of an implantable device for tendon regeneration

Purpose. A major drawback of current cartilage and intervertebral disc (IVD) tissue engineering is that human mesenchymal stem cells (MSCs) from osteoarthritic (OA) patients express high levels of type X collagen. Type X collagen is a marker of late stage chondrocyte hypertrophy, linked with endochondral ossification, which precedes bone formation. However, it has been shown that a novel plasma-polymer, called nitrogen-rich plasma-polymerized ethylene (PPE:N), is able to inhibit type X collagen expression in committed MSCs. The aim of this study was to determine if the decreased expression of type X collagen, induced by the PPE:N surfaces is maintained when MSCs are removed from the surface and transferred to pellet cultures in the presence of serum and growth factor free chondrogenic media. Method. Human MSCs were obtained from aspirates from the intramedullary canal of donors undergoing total hip replacement for OA. Cells were expanded for 2–3 passages and then cultured on polystyrene dishes and on two different PPE:N surfaces: high (H) and low (L) pressure deposition. Cells were transferred for 7 additional days in chondrogenic serum free media (DMEM high glucose supplemented with 2 mM L-glutamine, 20 mM HEPES, 45 mM NaHCO3, 100 U/ml penicillin, 100 ug/ml streptomycin, 1 mg/ml bovine serum albumin, 5 ug/ml insulin, 50 ug/ml ascorbic acid, 5 ng/ml sodium selenite, 5 ug/ml transferrin) in pellet culture or on PS cell culture dishes. RNA was extracted using a standard TRIzol protocol. RT-PCR was realized using Superscript II (RT) and Taq polymerase (PCR) with primers specific for type I and X collagen. GAPDH was used as a housekeeping gene and served to normalize the results. Results. As observed in previous studies, type X collagen mRNA level was suppressed when cultured on both H- and L-PPE:N. HPPE:N was more effective in decreasing type X collagen expression than LPPE:N (55 vs. 78 % of control OA cells). Results also showed that the decreased type X collagen mRNA level was maintained not only when cells were removed from the PPE:N surfaces and transferred to new polystyrene culture dishes in the presence of chondrogenic media, but also when transferred to pellet cultures. Culturing MSCs from OA patients on PPE:N surfaces and in pellet culture had however no effect on the level of type I collagen mRNA. Conclusion. The present study confirmed the potential of PPE:N surfaces in suppressing type X collagen expression in MSCs from OA patients. More importantly, when these cells are transferred to pellet cultures, type X collagen suppression is maintained. These results may lead us one step closer to the production of large amounts of reprogrammed MSCs for tissue engineering applications

Bone is capable of regeneration, and defects often heal spontaneously. However, cartilage, tendon, and ligament injuries usually result in replacement if the site by organized scar tissue, which is inferior to the native tissue. The osteogenic potential of mesenchymal stem cells (MSCs) has already been verified. MSCs hold great potential for the development of new treatment strategies for a host of orthopedic conditions. The multi-lineage potential and plasticity of MSCs allow them to be building blocks for a host of nonhematopoietic tissues, including bone. More recently, several groups have reported on the successful clinical application of tissue engineering strategies in the repair of bony defects in patients secondary to trauma and tumor resection. Advances in fabrication of biodegradable scaffolds that serve as beds for MSC implantation will hopefully lead to better biocompatibility and host tissue integration. Current strategies for bone tissue engineering include the use of osteoconductive matrix devices that promote bony ingrowth, and the delivery of osteoinductive growth factors, including bone morphogenetic protein (BMP) family, BMP-2 and BMP-7, to bony defect sites. Minimal toxicity has been observed in animal models involving genetically-manipulated stem cells transduced with retroviral and adenoviral vectors. Gene therapy using stem cells as delivery vehicles is a powerful weapon that can be used in a plethora of clinical situations that would benefit from the osteoinductive, proliferative, and angiogenic effects of growth factors. With better understanding of the biology of stem cells in the future and with enhancement of technologies that are capable to influence, modify, and culture these cells, a new field of regenerative skeletal medicine may emerge

PURPOSE. Recently, in tissue engineering several methods using stem cells have been developed to repair chondral and osteochondral defects. Most of these methods rely on the use of scaffolds. Studies in the literature have demonstrated, first in animals and then in humans, that the use of mesenchymal stem cells withdrawn by several methods from adipose tissue allows to regenerate hyaline articular cartilage. In fact, it has been cleared that adipose-derived cells have multipotentiality equivalent to bone marrow-derived stem cells and that they can very easily and very quickly be isolated in large amounts enabling their immediate use in operating room for one-step cartilage repair techniques. The purpose of this study is to evaluate the therapeutic effect of adipose-derived stem cells on cartilage repair and present our experience in the treatment of knee cartilage defects by the novel AMIC REPAIR TECHNIQUE AUGMENTED by immersing the collagen scaffold with mesenchymal stem cells withdrawn from adipose tissue of the abdomen. MATERIALS AND METHODS. Fat tissue processing involves mechanical forces and does not mandatorily require any enzymatic or chemical treatment in order to obtain the regenerative cells from the lipoaspirate. In our study, mesenchymal adipose stem cells were obtained by non-enzymatic filtration or microfragmentation of lipoaspirates of the abdomen adipose tissue that enabled the separation of the stromal vascular fraction and were used in one-step reconstruction of knee cartilage defects by means of this new AUGMENTED AMIC TECHNIQUE. The focal defects underwent bone marrow stimulation microfractures, followed by coverage with collagen double layer resorbable membrane (Chondro-gide. TM. -Geistlich Pharma AG, Wolhusen, Switzerland) soaked in the cells obtained from fat in 18 patients, aged between 31 and 58 years, at the level of the left knee in 10 cases and in the right in eight, with follow-up ranging between 12 and 36 months. RESULTS: Surgical procedures have been completed without technical problems neither intraoperative or early postoperative complications. The evaluation scores (IKDC, KOOS and VAS) showed a significant improvement, more than 30%, at the initial 6 months follow-up and furtherly improved in the subsequent follow-ups. Also the control MRIs showed a progressive filling and maturation of the repair tissue of the defects. CONCLUSIONS. Since we are reporting a short and medium-term experience, it is not, of course, possible to provide conclusive assessment considerations on this technique, as the experience has to mature along with the progression of follow-ups. The simplicity together with the absence of intraoperative difficulties or immediate complications and the experience gained by other authors, first in animals and then in early clinical cases, makes it, however, possible to say that this can be considered one of the techniques to which resort for one-step treatment of cartilage defects in the knee because it improves patient's conditions and has the potential to regenerate hyaline-like cartilage. Future follow-up works will confirm the results

Introduction. Rotator cuff tears remain a problem, with massive tears having a failure rate of repair reported of up to 60%, despite advances in surgical techniques. Tissue engineering techniques offers the possibility of regenerating damaged tendon tissue to a pre-injury state. We explore these techniques by implanting two novel tendon augmentation grafts with use of platelet rich plasma (PRP) in sheep. Methods. A total of 24 sheep were operated on, with the infraspinatus being surgically cut from its attachment to the humeral head. Each tendon was repaired using suture anchors and an interpositional implant according to 4 groups: (1) Empty control, (2) Novel collagen fibre implant with PRP (3) A novel collagen sponge implant (4) and the collagen sponge with PRP. The sheep were killed at 12 weeks and the implant site harvested and its histology evaluated. Results. Our findings showed that these novel grafts were well integrated into the tissue, with minimal inflammatory response. However, as expected, the material had not yet completely broken down. Our initial findings suggest that the combination of PRP with the collagen sponge best enhanced the repair of the tendon. Conclusion. Tissue engineered collagen graft hold great potential for the repair of tendons

Introduction. Massive rotator cuff repairs have up to 60% failure rate and repair of a chronic repair can have up to 40% failure rate. With this in mind, new methodologies are being to being developed to overcome this problem. The use of tendon augmentation grafts is one of them. Prior attempts have shown equivocal or poorer outcomes to control repairs. Aims and objectives: The specific aim of these expereiments was to test how well ovine tendon cells would take to a specific biological augmentation graft (Ligamimetic), and wheter tissue engineering techniques would enhance this. Method. Tendon cells harvested from ovine tendons will be cultured, exposed to the tendon augmentation graft, and analysed to see how well it takes to the tendon cells. We have conducted a 21 day experiment, sampling at days 7, 14, and 21. The experiment will look in sheep tendon cells:1. Platelet rich plasma: A comparison of the effects of platelet rich plasma to cell adherence, cell proliferation, and collagen production. Mesenchymal stem cell: A comparison of the effects of mesenchymal stem cells to the material on cell adherence, cell proliferation, and collagen production. Results. Our results show that the ovine tendon cells do take to the tendon augmentation graft, and are able to proliferate. We will present DAPI stainings, DNA and collagen turnover, with Westin blotting results. Results show that the addition of PRP had increased the cell adherence, cell proliferation, and collagen production. These effects were not seen with the mesenchymal stem cells. Conclusion. These experiments have shown our novel collagen scaffold augmentation graft has allowed cells to proliferate on it. Tissue engineered techniques also enhance cell proliferation, and has the potential to enhance cell repair

Background. Replacing bone lost as a consequence of trauma or disease is a major challenge in the treatment of musculoskeletal disorders. Tissue engineering strategies seek to harness the potential of stem cells to regenerate lost or damaged tissue. Bone marrow aspirate (BMA) provides a promising autologous source of skeletal stem cells (SSCs) however, previous studies have demonstrated that the concentration of SSCs required for robust tissue regeneration is below levels present in iliac crest BMA, emphasising the need for cell enrichment strategies prior to clinical application. Aims. To develop a novel strategy to enrich skeletal stem cells (SSCs) from human BMA, clinically applicable for intra-operative orthopaedic use. Methods. Iliac crest BMA was purchased from commercial suppliers and femoral canal BMA was obtained with informed consent from older patients undergoing total hip replacement. 5 to 40ml of BMA was processed to obtain 2–8 fold volume reductions. SSC function was assessed by assays for fibroblastic colony-forming units (CFU-F). Cell viability and seeding efficiency of processed and unprocessed aspirates applied to allograft was assessed. Results. Iliac crest BMA from 15 patients was enriched for SSCs in a processing time of only 15 minutes. Femoral BMA from 15 patients in the elderly cohort was concentrated up to 5-fold with a corresponding enrichment of viable, functional SSCs as confirmed by flow cytometry, CFU-F assays and histological analysis. The SSC enrichment of bone marrow aspirate significantly enhanced cell seeding efficiency onto allograft confirming the utility of this approach for application to bone regeneration. Conclusion. The ability to rapidly enrich BMA demonstrates the potential of this strategy for intra-operative application to enhance bone healing. The development of this device offers immediate potential for clinical application to reduce morbidity in many scenarios associated with local bone stock loss. Further analysis in vivo is ongoing prior to clinical tests

Treatment of segmental bone loss remains a major challenge in orthopaedic surgery. This study evaluated the healing potential of a series of highly porous tissue engineering scaffolds with the current clinical gold standard. We compare healing of collagen-glycosaminoglycan (CG) and collagen micro-hydroxyapatite (CHA) scaffolds, with and without recombinant bone morphogenetic protein-2 (BMP2), with autogenous bone graft (ABG) in the healing of a 15mm rabbit radius defect, which were filled with either CG scaffold, CHA scaffold, CG-BMP2, CHA-BMP2 or ABG. Serial radiographs and micro-computed tomography (µCT) at six week radiographs demonstrated complete defect bridging with callus using CHA and CG-BMP2 while the CHA-BMP2 was already in an advanced state of healing with cortical remodeling. By sixteen weeks CHA, CG-BMP2 and ABG all had advanced healing with cortical remodeling while CHA-BMP2 had complete anatomic healing. Quantitative histomorphometry values demonstrated similarly high healing levels of healing in CHA, CG-BMP2 and ABG with highest overall values in the CHA-BMP2 group. Thus, treatment of a critical sized, weight bearing, rabbit radius defect with a CHA scaffold can result in full cortical bridging with medullary cavity development. In addition, a CHA-BMP2 combination can result in fully mature, anatomic healing. The use of an off-the-shelf CHA scaffold for direct surgical placement into a defect site may be an effective bone graft substitute in the treatment of skeletal defects. The ease of manufacture, storage and peri-operative preparation may offer an alternative to traditional strategies, as well as to more recent BMP2 devices. This study provides clear evidence that CHA scaffolds can perform as well as autogenous bone grafts and supports their use as a viable alternative. Where the use of BMP2 may be desirable, these materials provide an ideal delivery mechanism and using a very low (near physiological) dose, healing superior to autogenous graft may be achieved

Purpose. Limb regeneration as it occurs in amphibians has two basic requirements: a source of multipotent cells capable of generating various tissues, and reorganization of those cells to form the one and only pattern of tissue appropriate to restore the missing parts. In the current biomedical world, there is much work dedicated to tissue engineering and to the differentiation of stem cells into various mature cell types. Neither of these approaches however, will by themselves succeed in regenerating a complex structure such as a limb. In our lab, we decided to focus on the pattern organization side of the equation by testing the potential of mammalian limb bud tissue to change its positional identity, and to manipulate that potential. Method. We used mouse embryos for our mammalian model. Small groups of cells were transplanted from one region of the limb bud into another, and the resulting effect on the positional identity of those cells was assessed using molecular markers of the upper arm, forearm and hand. We knocked out a genetic regulator of cell fate named Ezh2 specifically in the limb bud to test its role in committing cells to a given positional identity along the proximodistal limb axis. Results. We found that, similar to what one would expect for regenerating amphibians, mouse cells in the limb bud can alter their identity from that destined to make a hand to one poised to contribute to an upper arm, and vice versa. However, the cells ability to reset their identity is gradually lost during early development. In Ezh2 mutant mice, this cellular plasticity is prolonged, indicating that Ezh2 regulates commitment to a given fate. Ezh2 functions epigenetically by altering access of transcription factors to DNA. Conclusion. There is inherent plasticity in the ability of early mammalian limb bud cells with regard to positional identity. This plasticity can be prolonged by the alteration of a single gene. Many genes related to Ezh2 are functional in regulating cell fate. Manipulation of this family of genes to facilitate pattern reorganisation among a source of stem cells capable of making every relevant tissue type is one potential approach to engineering organised tissue in mammals

Introduction. Problematic bone defects are encountered regularly in orthopaedic practice particularly in fracture non-union, revision hip and knee arthroplasty, following bone tumour excision and in spinal fusion surgery. At present the optimal source of graft to ‘fill’ these defects is autologous bone but this has significant drawbacks including harvest site morbidity and limited quantities. Bone marrow has been proposed as the main source of osteogenic stem cells for the tissue-engineered cell therapy approach to bone defect management. Such cells constitute a minute proportion of the total marrow cell population and their isolation and expansion is a time consuming and expensive strategy. In this study we investigated human bone marrow stem cells as a potential treatment of bone defect by looking at variability in patient osteogenic cell populations as a function of patient differences. We produced a model to predict which patients would be more suited to cell based therapies and propose possible methods for improving the quality of grafts. Methods. Bone marrow was harvested from 30 patients undergoing elective total hip replacement surgery in Musgrave Park Hospital, Belfast (12 males, 18 females, age range 52-82 years). The osteogenic stem cell fraction was cultured and subsequently analysed using colony forming efficiency assays, flow cytometry, fluorescence activated cell sorting and proteomics. Results. The number and proliferative capacity of osteogenic stem cells varied markedly between patients. Statistical analysis revealed significantly better osteogenic capacity in:. male patients. samples in which the growth hormone Fibroblastic Growth Factor-2 was added to culture medium. patients who used the cholesterol lowering agent simvastatin. Patient use of inhaled steroids and NSAIDs were found to have detrimental effects. A statistical model to predict marrow profiles based on these variables was produced. Conclusions. Stem cell based tissue engineering represents the future of the treatment of bone defect. This study provides evidence that inter-patient variability in marrow cell colony forming and proliferation ability can in some way be explained by patient associated factors. Using this knowledge, we can identify which patients would be best suited to this method of treatment and propose techniques for enhancement of their graft profiles

In the treatment of ligament injuries there has been much interest in the restoration of the actual ligament anatomy, and the extent to which the original enthesis may be re-established. This study therefore seeks to uncover new information on ligament microstructure and its insertion into bone. Five bovine medial collateral ligaments (MCL) and five ovine anterior cruciate ligaments (ACL) were used in this study. All ligaments were harvested with the femoral and tibial bony insertions still intact. The bone ends were clamped and the MCL stretched to about 10% strain while the ACL underwent a 90° twist. The entire ligament-bone system, under load, was fixed in 10% formalin solution for 12 hours, following which it was partially decalcified to facilitate microsectioning. Thin 30 ìm-thick sections of the ligament-bone interface and ligament midsubstance were obtained. Differential Interference Contrast (DIC) optical microscopy was used to image the ligament and bone microarchitecture in the prescribed states of strain. Fibre crimp patterns were examined for the prescribed loading condition and showed distinct sections of fibre recruitment. Transverse micro-imaging of the ligament showed a significant variation in the sub-bundle cross-sectional area, ranging from 100ìm to 800 ìm. Those bundles closer to the central long axis of the ligament were numerous and small, while moving towards the periphery, they were large and singular. Both classifications of entheses, direct and indirect, were observed in the MCL insertions into the femur and tibia respectively. Of interest was the indirect insertion where the macro-level view of the near parallel attachment of fibres to bone via the periosteum was revealed, at the microscale, to involve a gradually increasing orthogonal insertion of fibres. This unique transition occurred closer to the joint line. In the ACL the anterior-medial (AM) and posterior-lateral (PL) bundles were easily discernable. All insertions into bone for the ACL were of the direct type. Fibres were thus seen to transition through the four zones of gradual mineralization to bone. However the manner in which the AM and PL bundles insert into bone, and the lateral soft tissue transition between these two bundles, revealed a structural complexity that we believe is biomechanically significant. This ‘mechano-structural’ investigation, using novel imaging techniques, has provided new insights into the microstructure of the ligament bone system. The images presented from this study are aimed to aid new approaches for reconstruction, and provide a blue-print for the design of ligament-bone systems via tissue engineering