Introduction. Porous scaffolds for bone ingrowth have numerous applications, including correcting deformities in the foot and ankle. Various materials and shapes may be selected for bridging an osteotomy in a corrective procedure. This research explores the performance of commercially pure Titanium (CPTi) and Tantalum (Ta) porous scaffold materials for use in foot and ankle applications under simplified compression loading. Methods. Finite element analysis was performed to evaluate von Mises stress in 3 porous implant designs: 1) a CPTi foot and ankle implant (Fig 1) 2) a similar Ta implant (wedge angle = 5°) and 3) a similar Ta implant with an increased wedge angle of 20°. Properties were assigned per reported material and density specifications. Clinically relevant axial compressive load of 2.5X BW (2154 N) was applied through fixtures which conform to ASTM F2077–11. Compressive yield and fatigue strength was evaluated per ASTM F2077–11 to compare CPTi performance in design 1 to the Ta performance of design 3. Results. FEA results indicate peak stresses at fixture contact locations. Similar designs (CPTi design 1 and Ta design 2) resulted in similar von Mises stresses (Fig 1). Increasing the wedge angle (Ta design 3) increased stress by 15%. The static compressive yield strength of CPTi design 1 (20,560 N) was similar to the Ta design 3 (20,902 N), with yield manifesting as barreling and crushing of the components (Fig 2a). However, the fatigue strength of CPTi (6,000 N) was 40% lower than the Ta design 3 (9,500 N) (Fig 3). In both cases fracture initiated from regions of highest stress predicted in FEA. Fracture progression was not instantaneous and was characterized by an accumulation of damage (Fig 2b–c) leading to gross component fracture and loss of implant integrity. Discussion. FEA is a useful tool to determine stress variations and can be used to identify worst case within a material: in this case, a larger implant wedge angle leads to higher stresses. Additionally, FEA accurately predicted fracture initiation location. However, material selection plays a large role in porous implant performance: although FEA predicted higher stresses in a Ta component with a greater wedge angle than a similar sized CPTi component, static compressive strengths were nearly identical, and the Ta component had 58% higher fatigue strength. When selecting a material or geometry for an implant application, both FEA and static testing allow for rapid evaluation of designs. However, caution should be used in interpreting the results: the ultimate performance of an implant in-vivo will depend on its ability to maintain integrity over a long period of time, and should be characterized by dynamic testing

Introduction: Articular cartilage has limited capacity for regeneration. Tissue engineering strategies offer future hope for cartilage replacement and repair. In an attempt to mimic functional native cartilage for tissue repair, current research focuses on construct/implant designs that simulate an embryonic like microenvironment to promote cellular differentiation along a chondrogenic lineage. The aim of the present study was, for the first time, to illustrate the differences between human neonatal and adult chondrocytes along with bone marrow stromal cells (HBMSCs) to differentiate the factors that promote chondrogenesis and maintain functional homeostasis.

Material and Methods: Adult chondrocytes, neonatal chondrocytes and HBMSCs were cultured in monolayers for 1, 2 and 3 weeks in basal or chondrogenic media. Expression of transcription factor Sox9, Aggrecan (ACAN) and Collagen type II (COL2A)was compared via real time polymerase chain reaction (q-PCR). Alternatively, cells were seeded onto 3D PLGA scaffolds and cultured in vitro for 3 and 6 weeks in basal or chondrogenic media. Paraffin sections of the constructs were stained with Alcian blue/ Sirius red and expression of Collagen type II and Aggrecan was visualised via immunohistochemistry.

Results: For monolayer cultures of all three cell types, at week 1, expression of all three genes was down regulated in basal medium compared to levels in chondrogenic medium. By week 2, q-PCR revealed an increased expression of Col2A in chondroinduced neonatal chondrocytes compared to adult chondrocytes and HBMSCs. A steady increase in SOX9 expression was observed with time in all three cell types in chondrogenic medium. However, SOX9 expression in week 2 was higher for each cell type in basal medium compared with chondrogenic medium. ACAN expression by HBMSCs was greatly enhanced compared with that of neonatal and adult chondrocytes after 2 weeks in chondrogenic medium. By week 3, basal cultures of all cell types showed an overall lower level of gene expression compared with chondroinduced cells. 3D constructs revealed the formation of cartilage like tissue for all three cell types with the presence of a prominent superficial layer and middle zone in the chondroinduced constructs. A superficial layer was also observed in constructs cultured in basal media but there was no evidence of any other characteristic zones. A fibrous capsule had formed around the chondroinduced tissue by week 6. Thinnest capsules were observed for constructs seeded with neonatal cells, with thickest capsules in constructs seeded with HBMSCs. Immunohistochemistry revealed a greater presence of aggrecan and type II collagen in the chondroinduced constructs compared to those cultures in basal media.

Conclusion: This comparative study indicates a major difference between the microenvironment of human neonatal chondrocytes, adult chondrocytes and HBMSCs. The expression of high amounts of COL2A and ACAN (considered to be middle to late markers in chondrogenesis) in week 1 in neonatal chondrocytes indicates a difference in temporal gene expression during chondrogenesis or in maintaining cartilage homeostasis. The study provides potentially useful information to inform cell-based therapies for cartilage regeneration.

The skeleton is tuned for sensing and responding to mechanical forces: a global bone strain moves the extra-cellular fluid through the lacunocanalicular network of compact bone, so gene expression of osteocytes is mechanically modulated by extra cellular fluid flow shear stress. Several studies showed that shear stress modulates bone cells gene expression: in vitro mechanical stimulation impacts the levels of alkaline phosphatase, cAMP, intracellular calcium, NO, prostaglandin E2, c-fos, COX-2, osteopontin and osteocalcin. Aim of this study is to investigate the effect of shear stress on SAOS-2 human osteoblasts proliferation, bone matrix production and mineralization, using a biostable polyurethane as scaffold and a perfusion bioreactor.

Polyurethane scaffolds with an average porediameter of 624 micron were utilized. Scaffolds were sterilized and placed in to standard well-plates (condition A) and into a bioreactor with forced perfusion at a rate of 3 ml/min (condition B). Human osteosarcoma cell lineSAOS-2 was obtained from the ATCC and cultured in McCoy’s 5A modified medium. A suspension of 7′105 osteoblasts was added onto the top of each scaffold. Medium was changed every 3 days and sampled for osteopontin and-osteocalcin ELISA kits. After 16 days culture DNA and calcium contents were measured, light microscopy and SEM analysis were performed.

In condition B, in comparison to A, we observed 33% higher cells proliferation, 12.6-fold higher osteopontin secretion, 99.6-foldhigher osteocalcin secretion and 8-fold higher calcium deposition. Microscopy observations revealed that in condition A osteoblasts were few with thin discontinuous extracellular matrix; in contrast shear stress induced 3D modeling of cells and matrix organization, so several cells were in multilayer with highly developed matrix and no surfaces were cell free.

Statically cultured osteoblasts showed normal proliferation, but a very low matrix synthesis. Into bioreactor, which provides physiological levels of shear stress, the osteoblasts proliferated and showed increased metabolic activity.

Bone marrow-derived mesenchymal stromal stem cells (BMSCs) are a promising cell source for treating articular cartilage defects. Quality of cartilaginous repair tissue following BMSC transplantation has been shown to correlate with functional outcome. Therefore, tissue-engineering variables, such as cell expansion environment and seeding density of scaffolds, are currently under investigation. The objectives of this study were to demonstrate chondrogenic differentiation of BMSCs seeded within a collagen I scaffold following isolation and expansion in two-dimensional (2D) and three-dimensional (3D) environments, and assess the impact of seeding density on in vitro chondrogenesis. It was hypothesised that both expansion protocols would produce BMSCs capable of hyaline-like chondrogenesis with an optimal seeding density of 10 million cells/cm3.

Ovine BMSCs were isolated in a 2D environment by plastic adherence, expanded to passage two in flasks containing expansion medium, and seeded within collagen I scaffolds (6 mm diameter, 3.5 mm thickness and 0.115 ± 0.020 mm pore size; Integra LifeSciences Corp.) at densities of 50, 10, 5, 1, and 0.5 million BMSCs/cm3. For 3D isolation and expansion, bone marrow aspirates containing known quantities of mononucleated cells (BMNCs) were seeded on scaffolds at 50, 10, 5, 1, and 0.5 million BMNCs/cm3 and cultured in expansion medium for an equivalent duration to 2D expansion. All cell-scaffold constructs were differentiated in vitro in chondrogenic medium containing transforming growth factor-beta three for 21 days and assessed with RT-qPCR, safranin O staining, histological scoring using the Bern Score, collagen immunofluorescence, and glycosaminoglycan (GAG) quantification.

Two dimensional-expanded BMSCs seeded at all densities were capable of proteoglycan production and displayed increased expressions of aggrecan and collagen II mRNA relative to pre-differentiation controls. Collagen II deposition was apparent in scaffolds seeded at 0.5–10 million BMSCs/cm3. Chondrogenesis of 2D-expanded BMSCs was most pronounced in scaffolds seeded at 5–10 million BMSCs/cm3 based on aggrecan and collagen II mRNA, safranin O staining, Bern Score, total GAG, and GAG/DNA. For 3D-expanded BMSC-seeded scaffolds, increased aggrecan and collagen II mRNA expressions relative to controls were noted with all densities. Proteoglycan deposition was present in scaffolds seeded at 0.5–50 million BMNCs/cm3, while collagen II deposition occurred in scaffolds seeded at 10–50 million BMNCs/cm3. The highest levels of aggrecan and collagen II mRNA, Bern Score, total GAG, and GAG/DNA occurred with seeding at 50 million BMNCs/cm3.

Within a collagen I scaffold, 2D- and 3D-expanded BMSCs are capable of hyaline-like chondrogenesis with optimal cell seeding densities of 5–10 million BMSCs/cm3 and 50 million BMNCs/cm3, respectively. Accordingly, these densities could be considered when seeding collagen I scaffolds in BMSC transplantation protocols.

Critical-sized bone defects remain challenging in the clinical setting. Autologous bone grafting remains preferred by clinicians. However, the use of autologous tissue is associated with donor-site morbidity and limited accessibility to the graft tissue. Advances in the development of synthetic bone substitutes focus on improving their osteoinductive properties. Whereas osteoinductivity has been demonstrated with ceramics, it is still a challenge in case of polymeric composites. One of the approaches to improve the regenerative properties of biomaterials, without changing their synthetic character, is the addition of inorganic ions with known osteogenic and angiogenic properties. We have previously reported that the use of a bioactive composite with high ceramic content composed of poly(ethyleneoxide terephthalate)/poly(butylene terephthalate) (1000PEOT70PBT30, PolyActive, PA) and 50% beta-tricalcium phosphate (β-TCP) with the addition of zinc in a form of a coating of the TCP particles can enhance the osteogenic differentiation of human mesenchymal stromal cells (hMSCs) (3). To further support the regenerative properties of these scaffolds, inorganic ions with known angiogenic properties, copper or cobalt, were added to the coating solution. β-TCP particles were immersed in a zinc and copper or zinc and cobalt solution with a concentration of 15 or 45 mM. 3D porous scaffolds composed of 1000PEOT70PBT30 and pure or coated β-TCP were additively manufactured by 3D fibre deposition. The osteogenic and angiogenic properties of the fabricated scaffolds were tested in vitro through culture with hMSCs and human umbilical vein endothelial cells, respectively. The materials were further evaluated through ectopic implantation in an in vivo mini-pig model. The early expression of relevant osteogenic gene markers (collagen-1, osteocalcin) of hMSCs was upregulated in the presence of lower concentration of inorganic ions. Further analysis will focus on the evaluation of ectopic bone formation and vascularisation of these scaffolds after implantation in a mini-pig ectopic intramuscular model

The regenerative capacity of hyaline cartilage is greatly limited. To prevent the onset of osteoarthritis, cartilage defects have to be properly treated. Cartilage, tissue engineered by mean of bioactive glass (BG) scaffolds presents a promising approach. Until now, conventional BGs have been used mostly for bone regeneration, as they are able to form a hydroxyapatite (HA) layer and are therefore, less suited for cartilage reconstruction. The aim of this study is to compare two BGs based on a novel BG composition tailored specifically for cartilage (CAR12N) and patented by us with conventional BG (BG1393) with a similar topology. The highly porous scaffolds consisting of 100% BG (CAR12N, CAR12N with low Ca2+/Mg2+ and BG1393) were characterized and dynamically seeded with primary porcine articular chondrocytes (pACs) or primary human mesenchymal stem cells (hMSCs) for up to 21 days. Subsequently, cell viability, DNA and glycosaminoglycan contents, cartilage-specific gene and protein expression were evaluated. The manufacturing process led to a comparable high (over 80%) porosity in all scaffold variants. Ion release and pH profiles confirmed bioactivity for them. After both, 7 and 21 days, more than 60% of the total surfaces of all three glass scaffold variants was densely colonized by cells with a vitality rate of more than 80%. The GAG content was significantly higher in BG1393 colonized with pACs. In general, the GAG content was higher in pAC colonized scaffolds in comparison to those seeded with hMSCs. The gene expression of cartilage-specific collagen type II, aggrecan, SOX9 and FOXO1 could be detected in all scaffold variants, irrespectively whether seeded with pACs or hMSCs. Cartilage-specific ECM components could also be detected at the protein level. In conclusion, all three BGs allow the maintenance of the chondrogenic phenotype or chondrogenic differentiation of hMSCs and thus, they present a high potential for cartilage regeneration

The in vitro mimicking of bone microenvironment for the study of pathologies is a challenging field that requires the design of scaffolds with suitable morphological, structural and cytocompatible properties. During last years, 3D in vitro tumour models have been developed to reproduce mechanical, biochemical and structural bone microenvironment elements, allowing cells to behave as in vivo. In this work, gas foamed polyether urethane foams (PUF) and 3D printed thermoplastic polyether urethane (3DP-PU) designed with different patterns are proposed as scaffolds for in vitro model of bone tissue. Surface coatings for a biomimetic behaviour of the 3D scaffold models were also investigated. Morphological, chemico-physical, mechanical properties, and biological in vitro behaviour were investigated. PUFs for metastases investigation. The suitability of PUF as 3D in vitro model to study the interactions between bone tumour initiating cells and the bone microenvironment was investigated. PUF open porosity (>70%) appeared suitable to mimic trabecular bone structure. Human adipose derived stem cells (ADSC) were cultured and differentiated into osteoblast lineage on the PU foam, as confirmed by Alizarin Red staining and RT-PCR, thus offering a bone biomimetic microenvironment to the further co-culture with bone derived tumour-initiating cells (MCFS). Tumour aggregates were observed after three weeks of co-culture by e-cadherin staining and SEM; modification in CaP distribution was identified by SEM-EDX and associated to the presence of tumour cells. 3DP-PU as tumour bone model. 3D printed scaffolds have pores with a precise and regular geometry (0°-90°, 0°-45°-90°-135°, 0°-60°-120°). PU scaffold porosity evidenced values from 55 to 67%, values that belong to the porosity range of the trabecular bone tissue (30-90%). The compressive modulus varied between 2 and 4 MPa, depending on the printed pattern. Biomimetic nanostructured coating was performed on 0-90° 3DP-PU by Ionized Jet Deposition. Coatings had a submicrometric thickness, variable tuning deposition time, nanostructured surface morphology and biomimetic composition. Coating on 3DP-PU promoted cells colonization of the whole porous scaffolds, compared to the controls, where cells concentrated mostly on the outer layers. In conclusion, based on the obtained results, scaffolds with different geometries have been successfully produced. Morphological and structural properties of the scaffolds here presented are suitable for mimicking the bone tissue, in order to produce a 3D in vitro model useful for bone pathologies research

Objective. In the present study, we aimed to assess whether gelatin/β-tricalcium phosphate (β-TCP) composite porous scaffolds could be used as a local controlled release system for vancomycin. We also investigated the efficiency of the scaffolds in eliminating infections and repairing osteomyelitis defects in rabbits. Methods. The gelatin scaffolds containing differing amounts of of β-TCP (0%, 10%, 30% and 50%) were prepared for controlled release of vancomycin and were labelled G-TCP0, G-TCP1, G-TCP3 and G-TCP5, respectively. The Kirby-Bauer method was used to examine the release profile. Chronic osteomyelitis models of rabbits were established. After thorough debridement, the osteomyelitis defects were implanted with the scaffolds. Radiographs and histological examinations were carried out to investigate the efficiency of eliminating infections and repairing bone defects. Results. The prepared gelatin/β-TCP scaffolds exhibited a homogeneously interconnected 3D porous structure. The G-TCP0 scaffold exhibited the longest duration of vancomycin release with a release duration of eight weeks. With the increase of β-TCP contents, the release duration of the β-TCP-containing composite scaffolds was decreased. The complete release of vancomycin from the G-TCP5 scaffold was achieved within three weeks. In the treatment of osteomyelitis defects in rabbits, the G-TCP3 scaffold showed the most efficacious performance in eliminating infections and repairing bone defects. Conclusions. The composite scaffolds could achieve local therapeutic drug levels over an extended duration. The G-TCP3 scaffold possessed the optimal porosity, interconnection and controlled release performance. Therefore, this scaffold could potentially be used in the treatment of chronic osteomyelitis defects. Cite this article: J. Zhou, X. G. Zhou, J. W. Wang, H. Zhou, J. Dong. Treatment of osteomyelitis defects by a vancomycin-loaded gelatin/β-tricalcium phosphate composite scaffold. Bone Joint Res 2018;7:46–57. DOI: 10.1302/2046-3758.71.BJR-2017-0129.R2

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

Abstract. Objectives. Direct ink writing (DIW) has gained considerable attention in production of personalized medical implants. Laponite nanoclay is added in polycaprolactone (PCL) to improve printability and bioactivity for bone implants. The 3D structure of DIW printed PCL/Laponite products was qualitatively evaluated using micro-CT. Methods. PCL/LP composite ink was formulated by dissolving 50% m/v PCL in dichloromethane with Laponite loading of up to 30%. The rheological properties of the inks were determined using Discovery HR-2 rheometer. A custom-made direct ink writer was used to fabricate both porous scaffold with 0°/90° lay-down pattern, and solid dumbbell-shaped specimens (ASTM D638 Type IV) with two printing orientations, 0° and 90° to the loading direction in tensile testing. The 3D structure of specimens was assessed using a micro-CT. Independent t-tests were performed with significance level at p<0.05. Results. The addition of Laponite in PCL ink has significantly enhanced viscosity for shape fidelity and shear-thinning property facilitating extrusion for DIW. Uniform distribution of Laponite was illustrated by micro-CT. For the 32-layer scaffold, interconnectivity of pores is observed at all 3 planes. The variation of height and width of layers is within 6% except the bottom 2 layers which are significantly lower and wider than other layers for mechanical support. For solid specimens, no ditches/interfaces between filaments are observed in 90° orientation while they are distinctive in 0° orientation because deposited filaments contact each other sooner in 90° orientation. 90° specimens also have lower air gap fraction (0.8 vs 5.4 %) and significantly higher Young's modulus (235 vs 195 MPa) and tensile strength (12.0 vs 9.5 MPa). Conclusions. The mechanical properties and printability of PCL/Laponite composites can be improved by controlling printing parameters; Micro-CT is an important tool to investigate the structure and properties of 3D printed products for bone tissue engineering

One of the latest trends in the field of tissue engineering is the development of in vitro 3D systems mimicking the target tissue or organ and thus recapitulating the tridimensional structure and microenvironment experienced by cells in vivo. Interestingly, certain tissues are known to be regulated by endogenous bioelectrical cues, in addition to chemical and mechanical cues. One such tissue is the bone. It has, indeed, been demonstrated to exhibit piezoelectric properties in vivo, with electrical signaling playing a role in its formation during the early embryo developmental stages. Electrical stimulation has been proven to sustain cell proliferation and to boost the expression of relevant genes and induce higher levels of enzymatic activities related to bone matrix deposition. Herein, we describe the development of a 3D model of bone tissue based on the conductive polymer PEDOT:PSS and human adipose derived stem cells. 3D electroactive porous scaffolds have been produced using the ice-templating technique, and different compositions (different ratios of conductive polymer to Collagen Type 1) have been explored. The developed scaffolds as well as cells interaction and response have been characterized. Overall, the results obtained so far highlight the usefulness of the porous conductive scaffolds as an in vitro platform for the development of 3D models for bone tissue engineering

Calcium phosphates-based coatings have been widely studied to favour a firm bonding between orthopaedic implants and the host bone. To this aim, thin films (thickness below 1 μm) having high adhesion to the substrate and a nanostructured surface texture are desired, capable of boosting platelet, proteins and cells adhesion. In addition, a tunable composition is required to resemble as closely as possible the composition of mineralized tissues and/or to intentionally substitute ions having possible therapeutic functions. The authors demonstrated nanostructured films having high surface roughness and a composition perfectly resembling the deposition target one can be achieved by Ionized Jet Deposition (IJD). Highly adhesive nanostructured coatings were obtained by depositing bone-apatite like thin films by ablation of deproteinized bovine bone, capable of promoting host cells attachment, proliferation and differentiation. Here, biomimetic films are deposited by IJD, using biogenic and synthetic apatite targets. Since IJD deposition can be carried out without heating the substrate, application on heat sensitive polymeric substrate, i.e. 3D printed porous scaffolds, is investigated. Biogenic apatite coatings are obtained by deposition of deproteinized bone (bovine, ovine, equine, porcine) and compared to ones of stoichiometry hydroxyapatite (HAp). Coatings composition (FT-IR-ATR, FT-IR microscopy, XRD, EDS) and morphology (SEM, AFM) are tested for deposition onto metallic and 3D-printed polymeric substrates (polyurethane (PU)). Different post-treatment annealing procedures for metallic substrates are compared (350–425°C), to optimize crystallinity. Then, uniformity of substrate coverage and possible damage caused to the polymeric substrate are studied by SEM, DSC and FT-IR microscopy. Biogenic coatings are composed by carbonated HAp (XRD, FT-IR). Trace ions Na. +. and Mg. 2+. are transferred from deposition target to coating. All coatings are nanostructured, composed by nano-sized globular aggregates, of which morphology and dimensions depend on the target characteristics. As-deposited coatings are amorphous, but crystallinity can be tuned by post-treatment annealing. A bone-like crystallinity can be achieved for heating at ≥400°C, also depending on duration. When deposited on 3D-printed PU scaffolds, coatings, owing to sub-micrometric thickness, coat them entirely, without altering their fibre shape and porosity. Obtained biomimetic bone apatite coatings can be deposited onto a variety of metallic and polymeric biomedical devices, thus finding several perspective applications in biomedical field

The aim of this study is to print 3D polycaprolactone (PCL) scaffolds at high and low temperature (HT/LT) combined with salt leaching to induced porosity/larger pore size and improve material degradation without compromising cellular activity of printed scaffolds. PCL solutions with sodium chloride (NaCl) particles either directly printed in LT or were casted, dried, and printed in HT followed by washing in deionized water (DI) to leach out the salt. Micro-Computed tomography (Micro-CT) and scanning electron microscope (SEM) were performed for morphological analysis. The effect of the porosity on the mechanical properties and degradation was evaluated by a tensile test and etching with NaOH, respectively. To evaluate cellular responses, human bone marrow-derived mesenchymal stem/stromal cells (hBMSCs) were cultured on the scaffolds and their viability, attachment, morphology, proliferation, and osteogenic differentiation were assessed. Micro-CT and SEM analysis showed that porosity induced by the salt leaching increased with increasing the salt content in HT, however no change was observed in LT. Structure thickness reduced with elevating NaCl content. Mass loss of scaffolds dramatically increased with elevated porosity in HT. Dog bone-shaped specimens with induced porosity exhibited higher ductility and toughness but less strength and stiffness under the tension in HT whereas they showed decrease in all mechanical properties in LT. All scaffolds showed excellent cytocompatibility. Cells were able to attach on the surface of the scaffolds and grow up to 14 days. Microscopy images of the seeded scaffolds showed substantial increase in the formation of extracellular matrix (ECM) network and elongation of the cells. The study demonstrated the ability of combining 3D printing and particulate leaching together to fabricate porous PCL scaffolds. The scaffolds were successfully printed with various salt content without negatively affecting cell responses. Printing porous thermoplastic polymer could be of great importance for temporary biocompatible implants in bone tissue engineering applications

Introduction. Bone tissue engineering approaches are an emerging strategy to treat bone defects, and commonly involve the delivery of osteogenic cells and/or drugs via a porous scaffold. We have been exploring an alternative injectable approach for drug delivery that would obviate the need for invasive surgery. Hypothesis. Sucrose Acetate Isobutyrate (SAIB) is a sucrose-based ester that is a highly viscous semi-solid. Diluting SAIB with 10–20% ethanol markedly reduces its viscosity, with ethanol diffusing rapidly after in vivo injection. This phase transitioning property makes SAIB an ideal candidate for bone tissue engineering. Materials and methods. The capacity of SAIB to act as a delivery system for recombinant human BMP-2 (rhBMP-2) was tested in a mouse ectopic bone formation model. In this model SAIB was used to deliver 0 to 10μg rhBMP-2. Next, SAIB was compared with porous collagen scaffold used clinically to delivery rhBMP-2 in a head-to-head trial. Commercial SAIB and SAIB produced in-house were also compared. Bone volumes were quantified by μCT. Discussion. Bone was found to form with as little as 2μg rhBMP-2 when delivered with SAIB. Injected SAIB also showed minimal inflammatory response and rapid breakdown, with bone formation occurring between one and two weeks. Conclusion. SAIB was found to be an effective delivery system for rhBMP-2 with translational utility. Future work will be required to examine the upscaling of this delivery system

All types of regenerative materials, including metal implants, porous scaffolds and cell-laden hydrogels, interact with the living tissue and cells. Such interaction is key to the settlement and regenerative outcomes of the biomaterials. Notably, the immune reactions from the host body crucially mediate the tissue-biomaterials interactions. Macrophages (as well as monocytes and neutrophils), traditionally best known as defenders, accumulate at the tissue-biomaterials interface and secrete abundant cytokines to create a microenvironment that benefits or inhibits regeneration. Because the phenotype of these cells is highly plastic in response to varying stimuli, it may be feasible to manipulate their activity at the interface and harness their power to mediate bone regeneration. Towards this goal, our team have been working on macrophage-driven bone regeneration in two aspects. First, targeting the abundant, glucan/mannan-recognising receptors on macrophages, we have devised a series of glucomannan polymers that can stimulate macrophages to secrete pro-osteogenic cytokines, and applied them as coating polymer of mesenchymal stem cells-laden hydrogels. Second, targeting the toll-like receptors (TLRs) on macrophages, we have screened TLR-activating polysaccharides and picked up zymosan (beta-glucan) to be modified onto titanium and glass implants. We evaluated both the efficacy of integration and safety of immune stimulation in both in vitro and in vivo models. Our future exploration lies in further elaborating the different roles and mechanisms of macrophages of various types and origins in the regenerative process

The ideal bone substituting biomaterials should possess bone-mimicking mechanical properties; have of porous interconnected structure, and adequate biodegradation behaviour to enable full recovery of bony defects. Direct metal printed porous scaffolds hold potential to satisfy all these requirements and were additively manufactured (AM) from atomized WE43 magnesium alloy powder with grain sizes between 20 and 60 μm. Their micro-structure, mechanical properties, degradation behavior and biocompatibility was then evaluated in vitro. Firstly, post-processing values nicely followed design parameters. Next, Young's moduli were similar to that of trabecular bone (i.e., E = 700–800 MPa) even after 28 days of simulated in vivo-like corrosion by in vitro immersion. Also, a relatively moderate hydrogen evolution, corresponding to a calculated 19.2% of scaffold mass loss, was in good agreement with 20.7% volume reduction as derived from reconstructed μCT images. Finally, only moderate cytotoxicity (i.e., level 0, <25%), even after extensive ISO 10993-conform testing for 72 h using MG-63 cells, was determined using WE43 extracts (2 way ANOVA, post-hoc Tukey's multiple comparisons test; α = 0.05). Cytotoxicity was further evaluated by direct live-dead staining assays, revealing a higher cell death in static culture. However, intimate cell-metal contact was observed by SEM. In summary, while pure WE43 may not yet be an ideal surface for cell adhesion, this novel AM process allows for adjusting biodegradation through topological design. Our approach holds tremendous potential to develop functional and biodegradable implants for orthopaedic applications

Introduction: The treatment of bone defects that occurs following fractures, the excision of bone tumours and at revision arthroplasty surgery, often involves the use of either autologous or allogenous bone grafts. However, both grafts have limitations. The aim of tissue engineering is to produce cells within an extracellular matrix that resembles tissue, which can be implanted into a patient to heal a tissue defect. The potential to engineer bone tissue grafts from patients’ autologous cells would improve the treatment of bone defects. Bone marrow contains cells, known as mesenchymal stem cells (MSCs), which have the ability to differentiate into osteoblasts. To create a 3-dimensional structure necessary for the reconstruction of tissue, cells need to be grown on a scaffold, for which hydroxyapatite (HA) was used, as it is osteoconductive. In living bone, increased extravascular perfusion increases new bone formation. Thus, these physiological conditions were reproduced in our novel bioreactor by perfusing MSCs seeded on porous HA scaffolds at a rate of 6ml/hr. Hypotheses: 1. Culture in this bioreactor improves cell penetration through a HA scaffold. 2. MSCs cultured on HA in this bioreactor differentiated into osteoblasts. Method: MSCs were isolated from 8 bone marrow aspirates, which were taken from patients during orthopaedic procedures following informed consent. For each experiment, MSCs from each patient were seeded onto 2 x 1cm. 3. scaffolds. To test cell penetration, the HA scaffolds were cultured for 7 days, then sectioned longitudinally and the number of cells were counted at increasing depths. Observations of MSCs on HA were compared under scanning (SEM) and transmission (TEM) electron microscopy. The HA scaffolds were cultured with MSCs in the bioreactor for 5, 10 & 15 days, after which time alkaline phosphatase (ALP) and type I pro-collagen protein levels were measured. Results: Penetration of cells through the porous HA scaffold was significantly greater when the cells had been cultured in the bioreactor (P< 0.05). Observing MSCs after 7 days in bioreactor culture under SEM, adherent fibroblastic cells formed a network over the HA. However, by 14 days the HA was covered with cuboidal cells, consistent with osteoblasts. TEM results showed that MSCs cultured on HA in the bioreactor produced organised collagen matrix after 28 days. Osteoblastic protein levels were significantly greater at each time point when MSCs were cultured in bioreactor conditions: ALP (P< 0.005) and type I pro-collagen (P< 0.05). Discussion and Conclusions: These results show that when cultured in our novel bioreactor, MSCs penetrated uniformly through the porous HA scaffold, whereas few cells penetrated in static culture conditions. Thus, our bio-reactor significantly improves the 3-dimensional growth of cells, resembling tissue. Moreover, in this study MSCs grown on HA in the bioreactor produced significantly larger amounts of ALP and type I pro-collagen, indicating that the MSCs differentiated into osteoblasts. Observations under TEM showed extracellular collagen matrix production which, when mineralized, produces bone. Therefore, this culture method could potentially be used to convert MSCs, isolated from patients’ bone marrow, into tissue-engineered bone

Abstract. Objectives. Assess and characterise the suitability of a novel silk reinforced biphasic 3D printed scaffold for osteochondral tissue regeneration. Methods. Biphasic hybrid scaffolds consisted of 3D printed poly(ethylene glycol)-terephthalate-poly(butylene terephthalate)(PEGT/PBT) scaffold frame work (pore size 0.75mm), which has been infilled with a cast and freeze dried porous silk scaffold (5×5×2mm. 3. ), in addition to a seamless silk top layer (1mm). Silk scaffolds alone were used as controls. Both the biphasic and control scaffolds were characterised via uniaxial compression testing (strain rate 0.1mm/min), and the potential biocompatibility of the scaffolds was tested via in vitro culture of seeded bone marrow stromal cells post fabrication. Results. Uniaxial compression testing showed that the biphasic scaffolds (N=4) initially demonstrated similar behaviour on a stress-strain curve to a silk scaffold alone control group (N=6), until a strain of 30% was reached. After 30% strain, load was transitioned from the silk only chondral layer to the 3D printed PEGT/PBT scaffold which resisted further compression and exhibited a significantly greater compressive modulus of 12.6±0.9MPa compared to 0.113±0.01MPa (p<0.001) in the silk scaffold control group. Following 24hours of seeding, no difference was noticed in cell adhesion behaviour under fluorescent microscopy between silk scaffolds and biphasic scaffolds (n=5). Discussion. The use of 3D printing within this novel scaffold provides a solid framework and increases its versatility. The reinforced silk not only provides the secondary Porous structure to the 3D printed scaffold for the bone phase, but also a superficial layer for the cartilage phase. This unique structure has the potential to fill a niche within osteochondral tissue regeneration, especially with the possibility for its use within personalised medicine. Conclusions. These results demonstrate that the novel silk reinforced biphasic 3D printed scaffold is biocompatible and has suitable mechanical properties for osteochondral tissue regeneration. Declaration of Interest. (b) declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research reported:I declare that there is no conflict of interest that could be perceived as prejudicing the impartiality of the research project

Due to its avascular nature, articular cartilage exhibits a very limited capacity to regenerate and to repair. Although much of the engineered cartilage grafts so far proposed have successfully shown to mimic the morphological and biochemical appearance of hyaline cartilage, they are generally mechanically inferior to the natural tissue. 1. In this study a new bioreactor device was realized to test innovative scaffolds under physiological stimulation (i.e. perfusion fluid flow and dynamic compression), with the aim to produce a more functional engineered tissue construct for articular applications. The computer-controlled bioreactor system has been properly designed to simultaneously provide static or dynamic compression and/or continuous perfusion to 3D engineered constructs, reproducing the physiological loads to which the articular cartilage is subjected. The specifically designed bioreactor comprises a chamber where the grafts are accommodated, a porous piston connected to a linear stepper motor (Dings, Model 34-2080-4-300), which controls its movement to provide mechanical stimulation and a peristaltic pump (Watson-Marlow, Model 323S), connected by joints and pipes to the culture chamber to ensure a continuous media perfusion. As piston for compression, a sintered stainless steel filter (43% of porosity) was adopted to allow the perfusion of the culture media during physical stimulation. The culture chamber is composed by a hollow cylinder (30 mm × 40,5 mm) and a base realized as a single object. They are made in polycarbonate for its characteristics of transparency and infrangibility and linked to a Nylon cover through four brass tie rods unscrewable from above. The chamber has been designed to accommodate simultaneously different constructs of any size and shape and stimulate them with perfusion and/or dynamic compression. A finites elements program was used to mimic the effects of perfusion and compression regime on the scaffolds cultured within the bioreactor chamber. The bioreactor was properly designed and developed. Particular attention has been paid to the implementation of a simple, compact and economical system. It was then tested by using different polymeric porous scaffolds (PVA, collagen, Gelatin grafts, both porous and not) cultured with mesenchymal cells up to two weeks. The system has been validated in terms of sterility, experimental reproducibility and ease to use. The structural stability of grafts over time has been observed; moreover cells adhesion, proliferation and matrix production under different chemical-physical stimuli conditions is under investigation. We have realized a novel bioreactor system representing an artificial articular niche, where a dynamic compression combined with fluid perfusion allows to functionally and mechanically validate tissue substitutes, besides investigating the response of engineered cartilaginous tissues to physical stimuli mimicking the natural cartilage micro-environment. Such bioreactor may be in fact adopted as a sort of articular simulator for promoting and standardizing the new tissue formation in vitro, preconditioning cell fate through the application of proper artificial stimuli. Moreover, they can be valid tools to investigate physiological processes and novel therapeutic approaches avoiding controversial animal models

Addressing bone defects is a complex medical challenge that involves dealing with various skeletal conditions, including fractures, osteoporosis (OP), bone tumours, and bone infection defects. Despite the availability of multiple conventional treatments for these skeletal conditions, numerous limitations and unresolved issues persist. As a solution, advancements in biomedical materials have recently resulted in novel therapeutic concepts. As an emerging biomaterial for bone defect treatment, graphene oxide (GO) in particular has gained substantial attention from researchers due to its potential applications and prospects. In other words, GO scaffolds have demonstrated remarkable potential for bone defect treatment. Furthermore, GO-loaded biomaterials can promote osteoblast adhesion, proliferation, and differentiation while stimulating bone matrix deposition and formation. Given their favourable biocompatibility and osteoinductive capabilities, these materials offer a novel therapeutic avenue for bone tissue regeneration and repair. This comprehensive review systematically outlines GO scaffolds’ diverse roles and potential applications in bone defect treatment.

Cite this article: Bone Joint Res 2024;13(12):725–740.