An endochondral ossification approach to early stage bone repair: Use of tissue-engineered hypertrophic cartilage constructs as primordial templates for weight-bearing bone repair.

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.

Controlled release of vascular endothelial growth factor from spray-dried alginate microparticles in collagen-hydroxyapatite scaffolds for promoting vascularization and bone repair.

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.

Content-Dependent Osteogenic Response of Nanohydroxyapatite: An in Vitro and in Vivo Assessment within Collagen-Based Scaffolds.

The use of collagen-based scaffolds in orthopedic applications has been limited due to poor mechanical properties, but this may be overcome by the introduction of a stiffer supporting phase. Thus, we developed a synthesis technique to produce nonaggregating, stable nanohydroxyapatite (nHA) particles, permitting the fabrication of biomimetic-inspired scaffolds through the combination of nanosized HA with collagen, as found in native bone. This study evaluates the mechanical and biological impact of incorporating increasing concentrations of these nanoparticles into porous collagen scaffolds (1:1 and 5:1 weight ratios of nHA/collagen). Mechanical assessment demonstrated that increasing nHA incorporation correlated with increasing Young's moduli, which could be further amplified using cross-linking treatments. Typically, the porosity of a scaffold is sacrificed to produce a stiffer material; however, through the use of nanosized particles the inclusion of up to 5:1 nHA/collagen content still preserved the high 99% porosity of the composite scaffold, allowing for maximum cell infiltration. Moreover, increasing nHA presence induced significant bioactive responses, achieving superior cellular attachment and enhanced osteogenesis, promoting earlier expression of bone markers and cell-mediated mineralization versus nHA-free collagen controls. Interestingly, these content-dependent results observed in vitro did not directly translate in vivo. Instead, similar levels of bone formation were achieved within critical-sized rat calvarial defects, independent of nHA content, following acellular implantation. The addition of nHA, both 1:1 and 5:1, induced significantly higher levels of mineralization and de novo bone ingrowth versus collagen controls as demonstrated by microcomputed tomography, histological, and histomorphometric analyses. Ultimately, these results demonstrate the immense osteoinductivity of nonaggregated nanoparticles of HA incorporated into collagen-composite scaffolds and emphasize the importance of in vivo-based evaluation of therapies intended for clinical use.

An Endochondral Ossification-Based Approach to Bone Repair: Chondrogenically Primed Mesenchymal Stem Cell-Laden Scaffolds Support Greater Repair of Critical-Sized Cranial Defects Than Osteogenically Stimulated Constructs In Vivo.

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 < 0.05) within critical-sized bone defects 8 weeks postimplantation. We speculate this to be associated with the secretion of angiogenic signals as demonstrated by the higher VEGF protein production in the ECO-based constructs before implantation leading to the greater blood vessel ingrowth. This study thus demonstrates the ability of recapitulating a developmental process of bone formation to develop tissue-engineered constructs that manifest appreciable promise for bone defect repair.

Functionalization of a Collagen-Hydroxyapatite Scaffold with Osteostatin to Facilitate Enhanced Bone Regeneration.

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.

Porous decellularized tissue engineered hypertrophic cartilage as a scaffold for large bone defect healing.

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.

Long-term controlled delivery of rhBMP-2 from collagen-hydroxyapatite scaffolds for superior bone tissue regeneration.

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.

Effect of different hydroxyapatite incorporation methods on the structural and biological properties of porous collagen scaffolds for bone repair.

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.

Recapitulating endochondral ossification: a promising route to in vivo bone 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.