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Mobilizing endogenous progenitor cells to repair damaged tissue in situ has the potential to revolutionize the field of regenerative medicine, while the early establishment of a vascular network will ensure survival of newly generated tissue. In this study, a gene-activated scaffold containing a stromal derived factor 1α plasmid (pSDF1α), a pro-angiogenic gene that is also thought to be involved in the recruitment of mesenchymal stromal cells (MSCs) to sites of injury is described. It is shown that over-expression of SDF1α protein enhanced MSC recruitment and induced vessel-like structure formation by endothelial cells in vitro. When implanted subcutaneously, transcriptomic analysis reveals that endogenous MSCs are recruited and significant angiogenesis is stimulated. Just 1-week after implantation into a calvarial critical-sized bone defect, pSDF1α-activated scaffolds are recruited MSCs and rapidly activate angiogenic and osteogenic programs, upregulating Runx2, Dlx5, and Sp7. At the same time-point, pVEGF-activated scaffolds are recruited a variety of cell types, activating endochondral ossification. The early response induced by both scaffolds leads to complete bridging of the critical-sized bone defects within 4-weeks. The versatile cell-free gene-activated scaffold described in this study is capable of harnessing and enhancing the body's own regenerative capacity and has immense potential in a myriad of applications., (© 2024 The Author(s). Advanced Healthcare Materials published by Wiley‐VCH GmbH.)

In recent decades, an increasing number of tissue engineered bone grafts have been developed. However, expensive and laborious screenings in vivo are necessary to assess the safety and efficacy of their formulations. Rodents are the first choice for initial in vivo screens but their size limits the dimensions and number of the bone grafts that can be tested in orthotopic locations. Here, we report the development of a refined murine subcutaneous model for semi-orthotopic bone formation that allows the testing of up to four grafts per mouse one order of magnitude greater in volume than currently possible in mice. Crucially, these defects are also "critical size" and unable to heal within the timeframe of the study without intervention. The model is based on four bovine bone implants, ring-shaped, where the bone healing potential of distinct grafts can be evaluated in vivo. In this study we demonstrate that promotion and prevention of ossification can be assessed in our model. For this, we used a semi-automatic algorithm for longitudinal micro-CT image registration followed by histological analyses. Taken together, our data supports that this model is suitable as a platform for the real-time screening of bone formation, and provides the possibility to study bone resorption, osseointegration and vascularisation., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Increasingly, tissue engineering strategies such as the use of biomaterial scaffolds augmented with specific biological cues are being investigated to accelerate the regenerative process. For example, significant clinical challenges still exist in efficiently healing large bone defects which are above a critical size. Herein, we describe a cell-free, biocompatible and bioresorbable scaffold incorporating a novel star-polypeptide biomaterial as a gene vector. This gene-loaded scaffold can accelerate bone tissue repair in vivo in comparison to a scaffold alone at just four weeks post implantation in a critical sized bone defect. This is achieved via the in situ transfection of autologous host cells which migrate into the implanted collagen-based scaffold via gene-loaded, star-shaped poly(l-lysine) polypeptides (star-PLLs). In vitro, we demonstrate that star-PLL nanomaterials designed with 64 short poly(l-lysine) arms can be used to functionalise a range of collagen based scaffolds with a dual therapeutic cargo (pDual) of the bone-morphogenetic protein-2 plasmid (pBMP-2) and vascular endothelial growth factor plasmid (pVEGF). The versatility of this polymeric vector is highlighted in its ability to transfect Mesenchymal Stem Cells (MSCs) with both osteogenic and angiogenic transgenes in a 3D environment from a range of scaffolds with various macromolecular compositions. In vivo, we demonstrate that a bone-mimetic, collagen-hydroxyapatite scaffold functionalized with star-PLLs containing either 32- or 64- poly(l-lysine) arms can be used to successfully deliver this pDual cargo to autologous host cells. At the very early timepoint of just 4 weeks, we demonstrate the 64-star-PLL-pDual functionalised scaffold as a particularly efficient platform to accelerate bone tissue regeneration, with a 6-fold increase in new bone formation compared to a scaffold alone. Overall, this article describes for the first time the incorporation of novel star-polypeptide biomaterials carrying two therapeutic genes into a cell free scaffold which supports accelerated bone tissue formation in vivo.

Due to the limited regenerative capacity of cartilage, untreated joint defects can advance to more extensive degenerative conditions such as osteoarthritis. While some biomaterial-based tissue-engineered scaffolds have shown promise in treating such defects, no scaffold has been widely accepted by clinicians to date. Multi-layered natural polymer scaffolds that mimic native osteochondral tissue and facilitate the regeneration of both articular cartilage (AC) and subchondral bone (SCB) in spatially distinct regions have recently entered clinical use, while the transient localized delivery of growth factors and even therapeutic genes has also been proposed to better regulate and promote new tissue formation. Furthermore, new manufacturing methods such as 3D bioprinting have made it possible to precisely tailor scaffold micro-architectures and/or to control the spatial deposition of cells in requisite layers of an implant. In this way, natural and synthetic polymers can be combined to yield bioactive, yet mechanically robust, cell-laden scaffolds suitable for the osteochondral environment. This mini-review discusses recent advances in scaffolds for osteochondral repair, with particular focus on the role of natural polymers in providing regenerative templates for treatment of both AC and SCB in articular joint defects., (© 2020 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

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., (Copyright © 2018 John Wiley & Sons, Ltd.)

The clinical translation of bioactive scaffolds for the treatment of large segmental bone defects has remained a challenge due to safety and efficacy concerns as well as prohibitive costs. The design of an implantable, biocompatible and resorbable device, which can fill the defect space, allow for cell infiltration, differentiation and neovascularisation, while also recapitulating the natural repair process and inducing cells to lay down new bone tissue, would alleviate the problems with existing treatments. We have developed a gene-activated scaffold platform using a bone-mimicking collagen hydroxyapatite scaffold loaded with chitosan nanoparticles carrying genes encoding osteogenic (BMP-2) and angiogenic (VEGF) proteins. With a single treatment, protein expression by mesenchymal stem cells (MSCs) seeded onto the scaffold is sustained for up to 28 days and is functional in inducing MSC osteogenesis. The in vivo safety and efficacy of this gene-activated scaffold platform was demonstrated resulting in the successful transfection of host cells, abrogating the requirement for multiple procedures to isolate cells or ex vivo cell culture. Furthermore, the level of bone formation at the exceptionally early time-point of 28 days was comparable to that achieved following recombinant BMP-2 protein delivery after 8 weeks in vivo, without the adverse side effects and at a fraction of the cost. This naturally derived cell-free gene-activated scaffold thus represents a new 'off-the-shelf' product capable of accelerating bone repair in critical-sized bone defects., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

A major limitation with current tissue-engineering approaches is creating functionally vascularized constructs that can successfully integrate with the host; this often leads to implant failure, due to avascular necrosis. In order to overcome this, the objective of the present work was to develop a method to incorporate growth factor-eluting alginate microparticles (MPs) into freeze-dried, collagen-based scaffolds. A collagen-hydroxyapatite (CHA) scaffold, previously optimized for bone regeneration, was functionalized for the sustained delivery of an angiogenic growth factor, vascular endothelial growth factor (VEGF), with the aim of facilitating angiogenesis and enhancing bone regeneration. VEGF was initially encapsulated in alginate MPs by spray-drying, producing particles of < 10 µm in diameter. This process was found to effectively encapsulate and control VEGF release while maintaining its stability and bioactivity post-processing. These VEGF-MPs were then incorporated into CHA scaffolds, leading to homogeneous distribution throughout the interconnected scaffold pore structure. The scaffolds were capable of sustained release of bioactive VEGF for up to 35 days, which was proficient at increasing tubule formation by endothelial cells in vitro. When implanted in vivo in a rat calvarial defect model, this scaffold enhanced vessel formation, resulting in increased bone regeneration compared to empty-defect and VEGF-free scaffolds. This biologically functionalized scaffold, composed entirely of natural-based materials, may offer an ideal platform to promote angiogenesis and tissue regeneration. Copyright © 2015 John Wiley & Sons, Ltd., (Copyright © 2015 John Wiley & Sons, Ltd.)

Limitations associated with demineralised bone matrix and other grafting materials have motivated the development of alternative strategies to enhance the repair of large bone defects. The growth plate (GP) of developing limbs contain a plethora of growth factors and matrix cues which contribute to long bone growth, suggesting that biomaterials derived from its extracellular matrix (ECM) may be uniquely suited to promoting bone regeneration. The goal of this study was to generate porous scaffolds from decellularised GP ECM and to evaluate their ability to enhance host mediated bone regeneration following their implantation into critically-sized rat cranial defects. The scaffolds were first assessed by culturing with primary human macrophages, which demonstrated that decellularisation resulted in reduced IL-1β and IL-8 production. In vitro, GP derived scaffolds were found capable of supporting osteogenesis of mesenchymal stem cells via either an intramembranous or an endochondral pathway, demonstrating the intrinsic osteoinductivity of the biomaterial. Furthermore, upon implantation into cranial defects, GP derived scaffolds were observed to accelerate vessel in-growth, mineralisation and de novo bone formation. These results support the use of decellularised GP ECM as a scaffold for large bone defect regeneration.

Bone grafts are the second most transplanted materials worldwide at a global cost to healthcare systems valued over $30 billion every year. The influence of microRNAs in the regenerative capacity of stem cells offers vast therapeutic potential towards bone grafting; however their efficient delivery to the target site remains a major challenge. This study describes how the functionalisation of porous collagen-nanohydroxyapatite (nHA) scaffolds with miR-133a inhibiting complexes, delivered using non-viral nHA particles, enhanced human mesenchymal stem cell-mediated osteogenesis through the novel focus on a key activator of osteogenesis, Runx2. This study showed enhanced Runx2 and osteocalcin expression, as well as increased alkaline phosphatase activity and calcium deposition, thus demonstrating a further enhanced therapeutic potential of a biomaterial previously optimised for bone repair applications. The promising features of this platform offer potential for a myriad of applications beyond bone repair and tissue engineering, thus presenting a new paradigm for microRNA-based therapeutics.

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., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

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 < 0.05, a scaffold known to support osteogenesis), while enabling good cell attachment, and moderate mesenchymal stem cell (MSC)-mediated calcium production after 28 days' culture (2.1-fold, P < 0.05, and 1.3-fold, respectively, vs. CG scaffolds). However, coating of collagen scaffolds with a hydroxyapatite precipitate after freeze-drying (CpHA scaffolds) has been shown to be a highly effective method to increase the compressive modulus (26-fold vs. CG controls, P < 0.001) of scaffolds while maintaining a high porosity (~ 98%). The coating of the ligand-dense collagen structure results in a lower cell attachment level (P < 0.05), although it supported greater cell-mediated calcium production (P < 0.0001) compared with other scaffold variants after 28 days' culture. The comparatively good mechanical properties of these high porosity scaffolds is obtained partially through highly crosslinking the scaffolds with both a physical (DHT) and chemical (EDAC) crosslinking treatment. Control of scaffold microstructure was examined via alterations in freezing temperature. It was found that the addition of HA prior to freeze-drying generally reduced the pore size and so the CpHA scaffold fabrication method offered increased control over the resulting scaffolds microstructure. These findings will help guide future design considerations for composite biomaterials and demonstrate that the method of HA incorporation can have profound effects on the resulting scaffold structural and biological response., (© 2014 Anatomical Society.)

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., (Copyright © 2014 John Wiley & Sons, Ltd.)

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., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The spatiotemporally controlled delivery of the pro-osteogenic factor rhBMP-2 would overcome most of the severe secondary effects linked to the products delivering this protein for bone regeneration. With this in mind, the aim of the present work was to develop a controlled rhBMP-2 release system using collagen-hydroxyapatite (CHA) scaffolds, which had been previously optimized for bone regeneration, as delivery platforms to produce a device with enhanced capacity for bone repair. Spray-drying and emulsion techniques were used to encapsulate bioactive rhBMP-2 in alginate and PLGA microparticles, with a high encapsulation efficiency. After incorporation of these microparticles into the scaffolds, rhBMP-2 was delivered in a sustained fashion for up to 28days. When tested in vitro with osteoblasts, these eluting materials showed an enhanced pro-osteogenic effect. From these results, an optimal rhBMP-2 eluting scaffold composition was selected and implanted in critical-sized calvarial defects in a rat model, where it demonstrated an excellent healing capacity in vivo. These platforms have an immense potential in the field of tissue regeneration; by tuning the specific therapeutic molecule to the tissue of interest and by utilizing different collagen-based scaffolds, similar systems can be developed for enhancing the healing of a diverse range of tissues and organs., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Vascularization and bone repair are accelerated by a series of gene-activated scaffolds delivering both an angiogenic and an osteogenic gene. Stem cell-mediated osteogenesis in vitro, in addition to increased vascularization and bone repair by host cells in vivo, is enhanced using all systems while the use of the nanohydroxyapatite vector to deliver both genes markedly enhances bone healing., (© 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Bone tissue engineering has emerged as one of the leading fields in tissue engineering and regenerative medicine. The success of bone tissue engineering relies on understanding the interplay between progenitor cells, regulatory signals, and the biomaterials/scaffolds used to deliver them--otherwise known as the tissue engineering triad. This review will discuss the roles of these fundamental components with a specific focus on the interaction between cell behaviour and scaffold structural properties. In terms of scaffold architecture, recent work has shown that pore size can affect both cell attachment and cellular invasion. Moreover, different materials can exert different biomechanical forces, which can profoundly affect cellular differentiation and migration in a cell type specific manner. Understanding these interactions will be critical for enhancing the progress of bone tissue engineering towards clinical applications.

Gene therapy involves the introduction of foreign genetic material into cells in order exert a therapeutic effect. The application of gene therapy to the field of orthopaedic tissue engineering is extremely promising as the controlled release of therapeutic proteins such as bone morphogenetic proteins have been shown to stimulate bone repair. However, there are a number of drawbacks associated with viral and synthetic non-viral gene delivery approaches. One natural polymer which has generated interest as a gene delivery vector is chitosan. Chitosan is biodegradable, biocompatible and non-toxic. Much of the appeal of chitosan is due to the presence of primary amine groups in its repeating units which become protonated in acidic conditions. This property makes it a promising candidate for non-viral gene delivery. Chitosan-based vectors have been shown to transfect a number of cell types including human embryonic kidney cells (HEK293) and human cervical cancer cells (HeLa). Aside from its use in gene delivery, chitosan possesses a range of properties that show promise in tissue engineering applications; it is biodegradable, biocompatible, has anti-bacterial activity, and, its cationic nature allows for electrostatic interaction with glycosaminoglycans and other proteoglycans. It can be used to make nano- and microparticles, sponges, gels, membranes and porous scaffolds. Chitosan has also been shown to enhance mineral deposition during osteogenic differentiation of MSCs in vitro. The purpose of this review is to critically discuss the use of chitosan as a gene delivery vector with emphasis on its application in orthopedic tissue engineering.

In the context of producing enhanced therapeutics for regenerative medicine, our laboratory develops gene-activated matrices (GAMs) using non-viral gene therapy (GT) in combination with collagen-based scaffolds engineered specifically for tissue repair. Non-viral vectors have been referred to as a minority pursuit in GT but considering the concerns associated with viral vectors and as transient gene expression is such a key consideration, further research is clearly warranted for tissue engineering (TE) applications. Mesenchymal stem cells (MSCs) are well regarded for their capability in bone regeneration but as primary cells, they are difficult to transfect. We have recently optimised the non-viral vector, polyethyleneimine (PEI), to achieve high transfection efficiencies in MSCs. Subsequently, a series of PEI-based GAMs were developed using collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite (collagen-nHa) scaffolds whereby transgene expression was detected up to 21 d with the collagen-nHa scaffold providing the most prolonged expression. Moreover, all PEI-based GAMs contained a low plasmid DNA dose of 2 µg which is far below doses often required in previous GAMs. Having successfully developed these GAMs, the ephrinB2 gene has recently been incorporated to produce a novel therapeutic GAM for bone repair. Herein, we discuss our recent investigations in the development and application of non-viral GAMs.

The healing potential of scaffolds for tissue engineering can be enhanced by combining them with genes to produce gene-activated matrices (GAMs) for tissue regeneration. We examined the potential of using polyethyleneimine (PEI) as a vector for transfection of mesenchymal stem cells (MSCs) in monolayer culture and in 3D collagen-based GAMs. PEI-pDNA polyplexes were fabricated at a range of N/P ratios and their optimal transfection parameters (N/P 7 ratio, 2μg dose) and transfection efficiencies (30±8%) determined in monolayer culture. The polyplexes were then loaded onto collagen, collagen-glycosaminoglycan and collagen-nanohydroxyapatite scaffolds where gene expression was observed up to 21 days with a polyplex dose as low as 2μg. Transient expression profiles indicated that the GAMs act as a polyplex depot system whereby infiltrating cells become transfected over time as they migrate throughout the scaffold. The collagen-nHa GAM exhibited the most prolonged and elevated levels of transgene expression. This research has thus demonstrated that PEI is a highly efficient pDNA transfection agent for both MSC monolayer cultures and in the 3D GAM environment. By combining therapeutic gene therapy with highly engineered scaffolds, it is proposed that these GAMs might have immense capability to promote tissue regeneration., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Purpose: The aim of this experiment was to examine the capacity of collagen-glycosaminoglycan scaffolds, with or without mesenchymal stem cells, to satisfactorily repair a 5-mm rat calvarial defect., Methods: Fifty-five Wistar rats were used in the study. The defects were either left empty to serve as controls (n = 7) or filled with cell-free scaffolds (n = 11), cell-seeded scaffolds that were pre-cultured in standard culture medium (n = 13), cell-seeded scaffolds that were pre-cultured in osteoinductive factor-supplemented medium (n = 12) or particulate autogenous bone (n = 12). The animals were sacrificed at 12 weeks after surgery, and specimens were prepared for histomorphometric analysis. The linear bone healing and the bone area within the defect were measured., Results: Comparable results were obtained using cell-free collagen-glycosaminoglycan scaffolds and autogenous bone both in terms of linear bone healing (P < 0.986) and area of new bone (P < 0.846). While the test groups showed significantly more bone formation compared to the empty defect control group, the linear bone healing and area of new bone within the defect were significantly lower in the cell-seeded scaffolds than in the cell-free scaffolds. The results have demonstrated that a cell-free collagen-glycosaminoglycan scaffold is capable of repairing a 5-mm rat calvarial defect as effectively as autogenous bone and that seeding the scaffold with pre-cultured mesenchymal stem cells prior to implantation offered no beneficial effect and resulted in incomplete healing of the defect., Conclusions: The results thus suggest that the scaffold has immense potential for tissue repair showing favorable osteoconductive properties, biocompatibility and degradability.

One of the key challenges in tissue engineering is to understand the host response to scaffolds and engineered constructs. We present a study in which two collagen-based scaffolds developed for bone repair: a collagen-glycosaminoglycan (CG) and biomimetic collagen-calcium phosphate (CCP) scaffold, are evaluated in rat cranial defects, both cell-free and when cultured with MSCs prior to implantation. The results demonstrate that both cell-free scaffolds showed excellent healing relative to the empty defect controls and somewhat surprisingly, to the tissue engineered (MSC-seeded) constructs. Immunological analysis of the healing response showed higher M1 macrophage activity in the cell-seeded scaffolds. However, when the M2 macrophage response was analysed, both groups (MSC-seeded and non-seeded scaffolds) showed significant activity of these cells which are associated with an immunomodulatory and tissue remodelling response. Interestingly, the location of this response was confined to the construct periphery, where a capsule had formed, in the MSC-seeded groups as opposed to areas of new bone formation in the non-seeded groups. This suggests that matrix deposited by MSCs during in vitro culture may adversely affect healing by acting as a barrier to macrophage-led remodelling when implanted in vivo. This study thus improves our understanding of host response in bone tissue engineering., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

There is an enduring and unmet need for a bioactive, load-bearing tissue-engineering scaffold, which is biocompatible, biodegradable and capable of facilitating and promoting osteogenesis when implanted in vivo. This study set out to develop a biomimetic scaffold by incorporating osteoinductive hydroxyapatite (HA) particles into a highly porous and extremely biocompatible collagen-based scaffold developed within our laboratory over the last number of years to improve osteogenic performance. Specifically we investigated how the addition of discrete quantities of HA affected scaffold porosity, interconnectivity, mechanical properties, in vitro mineralisation and in vivo bone healing potential. The results show that the addition of HA up to a 200 weight percentage (wt%) relative to collagen content led to significantly increased scaffold stiffness and pore interconnectivity (approximately 10 fold) while achieving a scaffold porosity of 99%. In addition, this biomimetic collagen-HA scaffold exhibited significantly improved bioactivity, in vitro mineralisation after 28 days in culture, and in vivo healing of a critical-sized bone defect. These findings demonstrate the regenerative potential of these biodegradable scaffolds as viable bone graft substitute materials, comprised only of bone's natural constituent materials, and capable of promoting osteogenesis in vitro and in vivo repair of critical-sized bone defects.

Lyophilised collagen scaffolds have shown enormous potential in tissue engineering in a number of areas due to their excellent biological performance. However, they are limited for use in bone tissue engineering due to poor mechanical properties. This paper discusses the development of a calcium-phosphate coating for collagen scaffolds in order to improve their mechanical properties for bone tissue engineering. Pure collagen scaffolds produced in a lyophilization process were coated by immersing them in sodium ammonium hydrogen phosphate (NaNH(4)HPO(4)) followed by calcium chloride (CaCl(2)). The optimal immersing sequence, duration, as well as the optimal solution concentration which facilitated improved mechanical properties of the scaffolds was investigated. The influence of the coating on composition, structural and material properties was analysed. This investigation successfully developed a novel collagen/calcium-phosphate composite scaffold. An increase in the mechanical properties of the scaffolds from 0.3 kPa to up to 90 kPa was found relative to a pure collagen scaffold, while the porosity was maintained as high as 92%, indicating the potential of the scaffold for bone tissue engineering or as a bone graft substitute.