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The hierarchical structure and function of the brain offer a source of inspiration for the development of intelligent robots.

Personalized and adaptive control systems can improve the efficacy of assistive technologies in rehabilitation.

Nerve guidance conduits (NGCs) are sub-optimal for long-distance injuries with inflammation and poor vascularization related to poor axonal repair. This study used a multi-factorial approach to create an optimized biomaterial NGC to address each of these issues. Through stepwise optimization, a collagen-chondroitin-6-sulfate (Coll-CS) biomaterial was functionalized with extracellular matrix (ECM) components; fibronectin, laminin 1 and laminin 2 (FibL1L2) in specific ratios. A snap-cooled freeze-drying process was then developed with optimal pore architecture and alignment to guide axonal bridging. Culture of adult rat dorsal root ganglia on NGCs demonstrated significant improvements in inflammation, neurogenesis and angiogenesis in the specific Fib:L1:L2 ratio of 1:4:1. In clinically relevant, large 15 mm rat sciatic nerve defects, FibL1L2-NGCs demonstrated significant improvements in axonal density and angiogenesis compared to unmodified NGCs with functional equivalence to autografts. Therefore, a multiparameter ECM-driven strategy can significantly improve axonal repair across large defects, without exogenous cells or growth factors.

Mimicking endochondral ossification to engineer constructs offers a novel solution to overcoming the problems associated with poor vascularisation in bone repair. This can be achieved by harnessing the angiogenic potency of hypertrophic cartilage. In this study, we demonstrate that tissue-engineered hypertrophically primed cartilage constructs can be developed from collagen-based scaffolds cultured with mesenchymal stem cells. These constructs were subsequently implanted into femoral defects in rats. It was evident that the constructs could support enhanced early stage healing at 4 weeks of these weight-bearing femoral bone defects compared to untreated defects. This study demonstrates the value of combining knowledge of development biology and tissue engineering in a developmental engineering inspired approach to tissue repair.

Peripheral nerve injury presents significant therapeutic challenges for recovery of motor and sensory function in patients. Different clinical approaches exist but to date there has been no consensus on the most effective method of treatment. Here, we investigate a novel approach to peripheral nerve repair using olfactory derived stem (ONS) cells delivered in a biphasic collagen and laminin functionalized hyaluronic acid based nerve guidance conduit (NGC). Nerve regeneration was studied across a 10-mm sciatic nerve gap in Sprague Dawley rats. The effect of ONS cell loading of NGCs with or without nerve growth factor (NGF) supplementation on nerve repair was compared to a cell-free NGC across a variety of clinical, functional, electrophysiological, and morphologic parameters. Animals implanted with ONS cell loaded NGCs demonstrated improved clinical and electrophysiological outcomes compared to cell free NGC controls. The nerves regenerated across ONS cell loaded NGCs contained significantly more axons than cell-free NGCs. A return of the nocioceptive withdrawal reflex in ONS cell treated animals indicated an advanced repair stage at a relatively early time point of 8 weeks post implantation. The addition of NGF further improved the outcomes of the repair indicating the potential beneficial effect of a combined stem cell/growth factor treatment strategy delivered on NGCs.

Emily Mayhew, a historian within the Department of Bioengineering at Imperial College London, talks to Nature Materials about the advances that have been made in medicine and, in particular, prosthetics since World War I.

Introduction Identification of a suitable treatment for osteochondral repair presents a major challenge due to existing limitations and an urgent clinical need remains for an off-the-shelf, low cost, one-step approach. A biomimetic approach, where the biomaterial itself encourages cellular infiltration from the underlying bone marrow and provides physical and chemical cues to direct these cells to regenerate the damaged tissue, provides a potential solution. To meet this need, a multi-layer collagen-based osteochondral defect repair scaffold has been developed in our group. Aim The objective of this study was to assess the in vivo response to this scaffold and determine its ability to direct regenerative responses in each layer in order to repair osteochondral tissue in a critical-sized defect in a rabbit knee. Methods Multi-layer scaffolds, consisting of a bone layer composed of type I collagen (bovine source) and hydroxyapatite (HA), an intermediate layer composed of type I and type II collagen and HA; and a superficial layer composed of type I and type II collagen (porcine source) and hyaluronic acid (HyA), were implanted into critical size (3 × 5 mm) osteochondral defects created in the medial femoral condyle of the knee joint of New Zealand white rabbits and compared to an empty control group. Repair was assessed macroscopically, histologically and using micro-CT analysis at 12 weeks post implantation. Results Analysis of repair tissue demonstrated an enhanced macroscopic appearance in the multi-layer scaffold group compared to the empty group. In addition, diffuse host cellular infiltration in the scaffold group resulted in tissue regeneration with a zonal organisation, with repair of the subchondral bone, formation of an overlying cartilaginous layer and evidence of an intermediate tidemark. Conclusion These results demonstrate the potential of this biomimetic multi-layered scaffold to support and guide the host reparative response in the treatment of osteochondral defects. Statement of Significance Osteochondral defects, involving cartilage and the underlying subchondral bone, frequently occur in young active patients due to disease or injury. While some treatment options are available, success is limited and patients often eventually require joint replacement. To address this clinical need, a multi-layer collagen-based osteochondral defect repair scaffold designed to direct host-stem cell mediated tissue formation within each region, has been developed in our group. The present study investigates the in vivo response to this scaffold in a critical-sized defect in a rabbit knee. Results shows the scaffolds ability to guide the host reparative response leading to tissue regeneration with a zonal organisation, repair of the subchondral bone, formation of an overlying cartilaginous layer and evidence of an intermediate tidemark.

Articular cartilage is an incredibly tough tissue owing to its ability to withstand repetitive compressive stress throughout an individual’s lifetime. Conversely, its single greatest limitation is the inability to heal even the most minor injuries (Newman, 1998). Due to the absence of a blood supply, articular cartilage responds to damage poorly (Nelson et al., 2010; Bora et al., 1987). Consequently, this predisposes the joint to articular cartilage degeneration. The repair of damaged tissue using conventional therapies and approaches has been elusive thus far. However, the use of tissue engineered biomaterials has shown promise in cartilage defect repair.In this context, the aim of this thesis was to develop a collagen-glycosaminoglycan (CG) scaffold with optimised intrinsic physico-chemical properties that might induce mesenchymal stem cell (MSC) differentiation towards a chondrogenic lineage in vitro. In addition, the effect of environmental factors such as oxygen tension and soluble growth factors in further enhancing chondrogenesis within these highly porous CG scaffolds was investigated. CG scaffolds developed in our laboratory have shown the potential to support MSC chondrogenesis (Farrell et al., 2006). In this thesis it was evident that different GAGs in the scaffolds elicit distinct cellular responses. In particular, hyaluronic acid stimulated enhanced migration, accelerated chondrogenic gene expression and cartilage matrix production in comparison to chondroitin sulphate. This thesis demonstrated that scaffold mean pore size plays a significant role in cellular behaviour. In particular, scaffolds with larger mean pore sizes supported significantly greater chondrogenic gene expression and accumulation of synthesised cartilage matrix in comparison to scaffolds with small mean pore sizes. In addition to the composition and micro-structure, this thesis also demonstrated that scaffold mechanical properties influence the fate of MSCs. Compliant scaffolds stimulated greater MSC chondrogenic differentiation whilst the stiffest scaffolds stimulated MSC osteogenic differentiation in the absence of differentiation factors. This further highlights the importance of scaffold physical characteristics in modulating the behaviour of progenitor cells. This thesis also looked at the effect of environmental factors on MSC chondrogenic differentiation in the optimised porous collagen-hyaluronic acid (CHyA) scaffolds. Low oxygen environments stimulated greater MSC chondrogenic differentiation with short term exposure to hypoxia eliciting additional enhancement chondrogenesis compared to normoxia. In order to further improve the biofunctionality we developed a bioactive CHyA scaffold for the delivery of therapeutic biomolecules such as TGF-P3 in order to enhance the regenerative capacity of the scaffold. It was evident that CHyA scaffolds subsequently permitted controlled release of the growth factors. Furthermore, control over their release rates could be achieved through manipulation of scaffold degradation rates. This demonstrates the potential of using these scaffold-based systems for the delivery of chondro-inductive growth factors with great implications over local control of cellular behaviour. Collectively, this study has led to the development of a type of CG scaffold with optimised composition, micro-architecture and mechanical properties which has significant capacity to promote cartilage regeneration. In addition, this thesis highlights the potential of using these scaffolds as templates for the development of tissue engineered constructs through enhancement of MSC-mediated chondrogenesis with environmental factors.

Hypertrophic cartilaginous grafts can be engineered in vitro using bone marrow derived Mesenchymal Stem Cells (MSCs). When such engineered tissues are implanted in vivo they have been shown to induce bone formation by recapitulating aspects of the developmental process of endochondral ossification. Alginate, a naturally sourced and biocompatible hydrogel, offers an attractive 3D environment to facilitate the in vitro chondrogenesis of MSCs. Furthermore, such alginate hydrogels can potentially be used to engineer cartilage tissues of scale to promote endochondral bone regeneration in large bone defects. The aim of this study was to investigate the ability of chondrogenically-primed MSC-laden alginate hydrogels to induce healing in two distinct critically-sized defect models. Bone marrow derived MSCs were seeded into alginate hydrogels, chondrogenically primed in vitro in the presence of TGF-β3 and then implanted into either a critically-sized rat cranial or femoral defect. μCT analysis 4 weeks post-implantation revealed significantly higher levels of mineralization within the femoral defects treated with MSC-laden alginate hydrogels compared to untreated empty controls, with similar results observed within the cranial defects. However, any newly deposited bone was generated appositional to the alginate material, and occurred only superficially or where the alginate was seen to degrade. Alginate material was found to persist within both orthotopic locations 8 weeks post-implantation, with its slow rate of degradation appearing to prevent complete bone regeneration. In conclusion, while chondrogenically primed MSC–alginate constructs can act as templates to treat critically-sized defects within bones formed through either intramembranous or endochondral ossification, further optimization of the degradation kinetics of the hydrogel itself will be required to accelerate bone tissue deposition and facilitate complete regeneration of such defects.

Defects within bones caused by trauma and other pathological complications may often require the use of a range of therapeutics to facilitate tissue regeneration. A number of approaches have been widely utilized for the delivery of such therapeutics via physical encapsulation or chemical immobilization suggesting significant promise in the healing of bone defects. The study focuses on the chemical immobilization of osteostatin, a pentapeptide of the parathyroid hormone (PTHrP107-111), within a collagen-hydroxyapatite scaffold. The chemical attachment method via crosslinking supports as little as 4% release of the peptide from the scaffolds after 21 d whereas non-crosslinking leads to 100% of the peptide being released by as early as 4 d. In vitro characterization demonstrates that this cross-linking method of immobilization supports a pro-osteogenic effect on osteoblasts. Most importantly, when implanted in a critical-sized calvarial defect within a rat, these scaffolds promote significantly greater new bone volume and area compared to nonfunctionalized scaffolds (**p < 0.01) and an empty defect control (***p < 0.001). Collectively, this study suggests that such an approach of chemical immobilization offers greater spatiotemporal control over growth factors and can significantly modulate tissue regeneration. Such a system may be adopted for a range of different proteins and thus offers the potential for the treatment of various complex pathologies that require localized mediation of drug delivery.

The clinical utilization of recombinant human bone morphogenetic protein 2 (rhBMP-2) delivery systems for bone regeneration has been associated with very severe side effects, which are due to the non-controlled and non-targeted delivery of the growth factor from its collagen sponge carrier post-implantation which necessitates supraphysiological doses. However, rhBMP-2 presents outstanding regenerative properties and thus there is an unmet need for a biocompatible, fully resorbable delivery system for the controlled, targeted release of this protein. With this in mind, the purpose of this work was to design and develop a delivery system to release low rhBMP-2 doses from a collagen-hydroxyapatite (CHA) scaffold which had previously been optimized for bone regeneration and recently demonstrated significant healing in vivo. In order to enhance the potential for clinical translation by minimizing the design complexity and thus upscaling and regulatory hurdles of the device, a microparticle and chemical functionalization-free approach was chosen to fulfill this aim. RhBMP-2 was combined with a CHA scaffold using a lyophilization fabrication process to produce a highly porous CHA scaffold supporting the controlled release of the protein over the course of 21days while maintaining in vitro bioactivity as demonstrated by enhanced alkaline phosphatase activity and calcium production by preosteoblasts cultured on the scaffold. When implanted in vivo, these materials demonstrated increased levels of healing of critical-sized rat calvarial defects 8weeks post-implantation compared to an empty defect and unloaded CHA scaffold, without eliciting bone anomalies or adjacent bone resorption. These results demonstrate that it is possible to achieve bone regeneration using 30 times less rhBMP-2 than INFUSE®, the current clinical gold standard; thus, this work represents the first step of the development of a rhBMP-2 eluting material with immense clinical potential.

Scaffolds which aim to provide an optimised environment to regenerate bone tissue require a balance between mechanical properties and architecture known to be conducive to enable tissue regeneration, such as a high porosity and a suitable pore size. Using freeze‐dried collagen‐based scaffolds as an analogue of native ECM, we sought to improve the mechanical properties by incorporating hydroxyapatite (HA) in different ways while maintaining a pore architecture sufficient to allow cell infiltration, vascularisation and effective bone regeneration. Specifically we sought to elucidate the effect of different hydroxyapatite incorporation methods on the mechanical, morphological, and cellular response of the resultant collagen‐HA scaffolds. The results demonstrated that incorporating either micron‐sized (CHA scaffolds) or nano‐sized HA particles (CnHA scaffolds) prior to freeze‐drying resulted in moderate increases in stiffness (2.2‐fold and 6.2‐fold, respectively, vs. collagen‐glycosaminoglycan scaffolds, P

The objective of this study was to develop a scaffold derived from cartilaginous extracellular matrix (ECM) that could be used as a growth factor delivery system to promote chondrogenesis of stem cells. Dehydrothermal crosslinked scaffolds were fabricated using a slurry of homogenized porcine articular cartilage, which was then seeded with human infrapatellar-fat-pad-derived stem cells (FPSCs). It was found that these ECM-derived scaffolds promoted superior chondrogenesis of FPSCs when the constructs were additionally stimulated with transforming growth factor (TGF)-β3. Cell-mediated contraction of the scaffold was observed, which could be limited by the additional use of 1-ethyl-3-3dimethyl aminopropyl carbodiimide (EDAC) crosslinking without suppressing cartilage-specific matrix accumulation within the construct. To further validate the utility of the ECM-derived scaffold, we next compared its chondro-permissive properties to a biomimetic collagen-hyaluronic acid (HA) scaffold optimized for cartilage tissue engineering (TE) applications. The cartilage-ECM-derived scaffold supported at least comparable chondrogenesis to the collagen-HA scaffold, underwent less contraction and retained a greater proportion of synthesized sulfated glycosaminoglycans. Having developed a promising scaffold for TE, with superior chondrogenesis observed in the presence of exogenously supplied TGF-β3, the final phase of the study explored whether this scaffold could be used as a TGF-β3 delivery system to promote chondrogenesis of FPSCs. It was found that the majority of TGF-β3 that was loaded onto the scaffold was released in a controlled manner over the first 10days of culture, with comparable long-term chondrogenesis observed in these TGF-β3-loaded constructs compared to scaffolds where the TGF-β3 was continuously added to the media. The results of this study support the use of cartilage-ECM-derived scaffolds as a growth factor delivery system for use in articular cartilage regeneration.

Despite its natural healing potential, bone is unable to regenerate sufficient tissue within critical-sized defects, resulting in a non-union of bone ends. As a consequence, interventions are required to replace missing, damaged or diseased bone. Bone grafts have been widely employed for the repair of such critical-sized defects. However, the well-documented drawbacks associated with autografts, allografts and xenografts have motivated the development of alternative treatment options. Traditional tissue engineering strategies have typically attempted to direct in vitro bone-like matrix formation within scaffolds prior to implantation into bone defects, mimicking the embryological process of intramembranous ossification (IMO). Tissue-engineered constructs developed using this approach often fail once implanted, due to poor perfusion, leading to avascular necrosis and core degradation. As a result of such drawbacks, an alternative tissue engineering strategy, based on endochondral ossification (ECO), has begun to emerge, involving the use of in vitro tissue-engineered cartilage as a transient biomimetic template to facilitate bone formation within large defects. This is driven by the hypothesis that hypertrophic chondrocytes can secrete angiogenic and osteogenic factors, which play pivotal roles in both the vascularization of constructs in vivo and the deposition of a mineralized extracellular matrix, with resulting bone deposition. In this context, this review focuses on current strategies taken to recapitulate ECO, using a range of distinct cells, biomaterials and biochemical stimuli, in order to facilitate in vivo bone formation.

Articular cartilage is a unique tissue owing to its ability to withstand repetitive compressive stress throughout an individual’s lifetime. However, its major limitation is the inability to heal even the most minor injuries. There still remains an inherent lack of strategies that stimulate hyaline-like articular cartilage growth with appropriate functional properties. Recent scientific advances in tissue engineering have made significant steps towards development of constructs for articular cartilage repair. In particular, research has shown the potential of biomaterial physico-chemical properties significantly influencing the proliferation, differentiation and matrix deposition by progenitor cells. Accordingly, this highlights the potential of using such properties to direct the lineage towards which such cells follow. Moreover, the use of soluble growth factors to enhance the bioactivity and regenerative capacity of biomaterials has recently been adopted by researchers in the field of tissue engineering. In addition, gene therapy is a growing area that has found noteworthy use in tissue engineering partly due to the potential to overcome some drawbacks associated with current growth factor delivery systems. In this context, such advanced strategies in biomaterial science, cell-based and growth factor-based therapies that have been employed in the restoration and repair of damaged articular cartilage will be the focus of this review article.

The response of mesenchymal stem cells (MSCs) to a matrix largely depends on the composition as well as the extrinsic mechanical and morphological properties of the substrate to which they adhere to. Collagen–glycosaminoglycan (CG) scaffolds have been extensively used in a range of tissue engineering applications with great success. This is due in part to the presence of the glycosaminoglycans (GAGs) in complementing the biofunctionality of collagen. In this context, the overall goal of this study was to investigate the effect of two GAG types: chondroitin sulphate (CS) and hyaluronic acid (HyA) on the mechanical and morphological characteristics of collagen-based scaffolds and subsequently on the differentiation of rat MSCs in vitro . Morphological characterisation revealed that the incorporation of HyA resulted in a significant reduction in scaffold mean pore size ( 93.9 μ m) relative to collagen–CS (CCS) scaffolds ( 136.2 μ m). In addition, the collagen–HyA (CHyA) scaffolds exhibited greater levels of MSC infiltration in comparison to the CCS scaffolds. Moreover, these CHyA scaffolds showed significant acceleration of early stage gene expression of SOX-9 (approximately 60-fold higher, p 0.01 ) and collagen type II (approximately 35-fold higher, p 0.01 ) as well as cartilage matrix production (7-fold higher sGAG content) in comparison to CCS scaffolds by day 14. Combining their ability to stimulate MSC migration and chondrogenesis in vitro , these CHyA scaffolds show great potential as appropriate matrices for promoting cartilage tissue repair.

In stem cell biology, focus has recently turned to the influence of the intrinsic properties of the extracellular matrix (ECM), such as structural, composition and elasticity, on stem cell differentiation. Utilising collagen-glycosaminoglycan (CG) scaffolds as an analogue of the ECM, this study set out to determine the effect of scaffold stiffness and composition on naive mesenchymal stem cell (MSC) differentiation in the absence of differentiation supplements. Dehydrothermal (DHT) and 1-ethyl-3-3-dimethyl aminopropyl carbodiimide (EDAC) crosslinking treatments were used to produce three homogeneous CG scaffolds with the same composition but different stiffness values: 0.5, 1 and 1.5 kPa. In addition, the effect of scaffold composition on MSC differentiation was investigated by utilising two glycosaminoglycan (GAG) types: chondroitin sulphate (CS) and hyaluronic acid (HyA). Results demonstrated that scaffolds with the lowest stiffness (0.5 kPa) facilitated a significant up-regulation in SOX9 expression indicating that MSCs are directed towards a chondrogenic lineage in more compliant scaffolds. In contrast, the greatest level of RUNX2 expression was found in the stiffest scaffolds (1.5 kPa) indicating that MSCs are directed towards an osteogenic lineage in stiffer scaffolds. Furthermore, results demonstrated that the level of up-regulation of SOX9 was higher within the CHyA scaffolds in comparison to the CCS scaffolds indicating that hyaluronic acid further influences chondrogenic differentiation. In contrast, enhanced RUNX2 expression was observed in the CCS scaffolds in comparison to the CHyA scaffolds suggesting an osteogenic influence of chondroitin sulphate on MSC differentiation. In summary, this study demonstrates that, even in the absence of differentiation supplements, scaffold stiffness can direct the fate of MSCs, an effect that is further enhanced by the GAG type used within the CG scaffolds. These results have significant implications for the therapeutic uses of stem cells and enhance our understanding of the physical effects of the in vivo microenvironment on stem cell behaviour.

Bone regeneration can be accelerated by localized delivery of appropriate growth factors/biomolecules. Localized delivery can be achieved by a 2-level system: (i) incorporation of biomolecules within biodegradable particulate carriers (nanoparticles), and (ii) inclusion of such particulate carriers (nanoparticles) into suitable porous scaffolds. In this study, freeze-dried porous chitosan–gelatin scaffolds (CH–G: 1:2 ratio by weight) were embedded with various amounts of poly(lactide-co-glycolide) (PLGA) nanoparticles, precisely 16.6%, 33.3% and 66.6% (respect to CH–G weight). Scaffolds loaded with PLGA nanoparticles were subjected to physico-mechanical and biological characterizations including morphological analysis, swelling and dissolution tests, mechanical compression tests and cell viability tests. Results showed that incorporation of PLGA nanoparticles into porous crosslinked CH–G scaffolds: (i) changed the micro-architecture of the scaffolds in terms of mean pore diameter and pore size distribution, (ii) reduced the dissolution degree of the scaffolds, and (iii) increased the compressive modulus. On the other hand, the water uptake behavior of CH–G scaffolds containing PLGA nanoparticles significantly decreased. The incorporation of PLGA nanoparticles did not affect the biocompatibility of CH–G scaffolds.

The lack of success associated with the use of bone grafts has motivated the development of tissue engineering approaches for bone defect repair. However, the traditional tissue engineering approach of direct osteogenesis, mimicking the process of intramembranous ossification (IMO), leads to poor vascularization. In this study, we speculate that mimicking an endochondral ossification (ECO) approach may offer a solution by harnessing the potential of hypertrophic chondrocytes to secrete angiogenic signals that support vasculogenesis and enhance bone repair. We hypothesized that stimulation of mesenchymal stem cell (MSC) chondrogenesis and subsequent hypertrophy within collagen-based scaffolds would lead to improved vascularization and bone formation when implanted within a critical-sized bone defect in vivo. To produce ECO-based constructs, two distinct scaffolds, collagen-hyaluronic acid (CHyA) and collagen-hydroxyapatite (CHA), with proven potential for cartilage and bone repair, respectively, were cultured with MSCs initially in the presence of chondrogenic factors and subsequently supplemented with hypertrophic factors. To produce IMO-based constructs, CHA scaffolds were cultured with MSCs in the presence of osteogenic factors. These constructs were subsequently implanted into 7 mm calvarial defects on Fischer male rats for up to 8 weeks in vivo. The results demonstrated that IMO- and ECO-based constructs were capable of supporting enhanced bone repair compared to empty defects. However, it was clear that the scaffolds, which were previously shown to support the greatest cartilage formation in vitro (CHyA), led to the highest new bone formation (p

Clinical translation of tissue engineered therapeutics is hampered by the significant logistical and regulatory challenges associated with such products, prompting increased interest in the use of decellularized extracellular matrix (ECM) to enhance endogenous regeneration. Most bones develop and heal by endochondral ossification, the replacement of a hypertrophic cartilaginous intermediary with bone. The hypothesis of this study is that a porous scaffold derived from decellularized tissue engineered hypertrophic cartilage will retain the necessary signals to instruct host cells to accelerate endogenous bone regeneration. Cartilage tissue (CT) and hypertrophic cartilage tissue (HT) were engineered using human bone marrow derived mesenchymal stem cells, decellularized and the remaining ECM was freeze-dried to generate porous scaffolds. When implanted subcutaneously in nude mice, only the decellularized HT-derived scaffolds were found to induce vascularization and de novo mineral accumulation. Furthermore, when implanted into critically-sized femoral defects, full bridging was observed in half of the defects treated with HT scaffolds, while no evidence of such bridging was found in empty controls. Host cells which had migrated throughout the scaffold were capable of producing new bone tissue, in contrast to fibrous tissue formation within empty controls. These results demonstrate the capacity of decellularized engineered tissues as ‘off-the-shelf’ implants to promote tissue regeneration.

Incorporation of therapeutics in the form of growth factors within biomaterials can enhance their biofunctionality. Two methods of incorporating transforming growth factor-beta 3 within collagen-hyaluronic acid scaffolds are described, markedly improving mesenchymal stem cell-mediated chondrogenic differentiation and matrix production. Such scaffolds offer control over the release of therapeutics, demonstrating their potential for repair of complex chondral defects requiring additional stimuli.

Despite its natural healing potential, bone is unable to regenerate sufficient tissue within critical-sized defects, resulting in a non-union of bone ends. As a consequence, interventions are required to replace missing, damaged or diseased bone. Bone grafts have been widely employed for the repair of such critical-sized defects. However, the well-documented drawbacks associated with autografts, allografts and xenografts have motivated the development of alternative treatment options. Traditional tissue engineering strategies have typically attempted to direct in vitro bone-like matrix formation within scaffolds prior to implantation into bone defects, mimicking the embryological process of intramembranous ossification (IMO). Tissue-engineered constructs developed using this approach often fail once implanted, due to poor perfusion, leading to avascular necrosis and core degradation. As a result of such drawbacks, an alternative tissue engineering strategy, based on endochondral ossification (ECO), has begun to emerge, involving the use of in vitro tissue-engineered cartilage as a transient biomimetic template to facilitate bone formation within large defects. This is driven by the hypothesis that hypertrophic chondrocytes can secrete angiogenic and osteogenic factors, which play pivotal roles in both the vascularization of constructs in vivo and the deposition of a mineralized extracellular matrix, with resulting bone deposition. In this context, this review focuses on current strategies taken to recapitulate ECO, using a range of distinct cells, biomaterials and biochemical stimuli, in order to facilitate in vivo bone formation.

Cartilage and osteochondral defects pose a significant challenge in orthopedics. Tissue engineering has shown promise as a potential method for the treatment of such defects; however, a long-lasting repair strategy has yet to be realized. This study focuses on the development of a layered construct for osteochondral repair, fabricated through a novel "iterative layering" freeze-drying technique. The process involved repeated steps of layer addition followed by freeze-drying, enabling control over material composition, pore size and substrate stiffness in each region of the construct, while also achieving a seamlessly integrated layer structure. The novel construct developed mimics the inherent gradient structure of healthy osteochondral tissue: a bone layer composed of type I collagen and hydroxyapatite (HA), an intermediate layer composed of type I collagen, type II collagen and HA and a cartilaginous region composed of type I collagen, type II collagen and hyaluronic acid. The material properties were designed to provide the biological cues required to encourage infiltration of host cells from the bone marrow while the biomechanical properties were designed to provide an environment optimized to promote differentiation of these cells towards the required lineage in each region. This novel osteochondral graft was shown to have a seamlessly integrated layer structure, high levels of porosity (>97%), a homogeneous pore structure and a high degree of pore interconnectivity. Moreover, homogeneous cellular distribution throughout the entire construct was evident following in vitro culture, demonstrating the potential of this multi-layered scaffold as an advanced strategy for osteochondral defect repair.