John P. Gleeson

Mesenchymal stem cell fate is regulated by the composition and mechanical properties of Collagen-Glycosaminoglycan scaffolds

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.

Development of a biomimetic collagen-hydroxyapatite scaffold for bone tissue engineering using a SBF immersion technique.

The objective of this study was to develop a biomimetic, highly porous collagen-hydroxyapatite (HA) composite scaffold for bone tissue engineering (TE), combining the biological performance and the high porosity of a collagen scaffold with the high mechanical stiffness of a HA scaffold. Pure collagen scaffolds were produced using a lyophilization process and immersed in simulated body fluid (SBF) to provide a biomimetic coating. Pure collagen scaffolds served as a control. The mechanical, material, and structural properties of the scaffolds were analyzed and the biological performance of the scaffolds was evaluated by monitoring the cellular metabolic activity and cell number at 1, 2, and 7 days post seeding. The SBF-treated scaffolds exhibited a significantly increased stiffness compared to the pure collagen group (4-fold increase), while a highly interconnected structure (95%) was retained. FTIR indicated that the SBF coating exhibited similar characteristics to pure HA. Micro-CT showed a homogeneous distribution of HA. Scanning electron microscopy also indicated a mineralization of the collagen combined with a precipitation of HA onto the collagen. The excellent biological performance of the collagen scaffolds was maintained in the collagen-HA scaffolds as demonstrated from cellular metabolic activity and total cell number. This investigation has successfully developed a biomimetic collagen-HA composite scaffold. An increase in the mechanical properties combined with an excellent biological performance in vitro was observed, indicating the high potential of the scaffold for bone TE.

Addition of hyaluronic acid improves cellular infiltration and promotes early-stage chondrogenesis in a collagen-based scaffold for cartilage tissue engineering.

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 μm) relative to collagen-CS (CCS) scaffolds (136.2 μm). 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.

A collagen-hydroxyapatite scaffold allows for binding and co-delivery of recombinant bone morphogenetic proteins and bisphosphonates.

An emerging paradigm in orthopedics is that a bone-healing outcome is the product of the anabolic (bone-forming) and catabolic (bone-resorbing) outcomes. Recently, surgical and tissue engineering strategies have emerged that combine recombinant human bone morphogenetic proteins (rhBMPs) and bisphosphonates (BPs) in order to maximize anabolism and minimize catabolism. Collagen-based scaffolds that are the current surgical standard can bind rhBMPs, but not BPs. We hypothesized that a biomimetic collagen-hydroxyapatite (CHA) scaffold would bind both agents and produce superior in vivo outcomes. Consistent with this concept, in vitro elution studies utilizing rhBMP-2 ELISA assays and scintillation counting of (14)C-radiolabeled zoledronic acid (ZA) confirmed delayed release of both agents from the CHA scaffold. Next, scaffolds were tested for their capacity to form ectopic bone after surgical implantation into the rat hind limb. Using CHA, a significant 6-fold increase in bone volume was seen in rhBMP-2/ZA groups compared to rhBMP-2 alone, confirming the ability of ZA to enhance rhBMP-2 bone formation. CHA scaffolds were found to be capable of generating mineralized tissue in the absence of rhBMP-2. This study has implications for future clinical treatments of critical bone defects. It demonstrates the relative advantages of co-delivering anabolic and anti-catabolic agents using a multicomponent scaffold system.

Multi-layered collagen-based scaffolds for osteochondral defect repair in rabbits

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.

Scaffold mean pore size influences mesenchymal stem cell chondrogenic differentiation and matrix deposition.

Recent investigations into micro-architecture of scaffolds has revealed that mean pore sizes are cell-type specific and influence cellular shape, differentiation, and extracellular matrix secretion. In this context, the overall goal of this study was to investigate whether scaffold mean pore sizes affect mesenchymal stem cell initial attachment, chondrogenic gene expression, and cartilage-like matrix deposition. Collagen-hyaluronic acid (CHyA) scaffolds, recently developed in our laboratory for in vitro chondrogenesis, were fabricated with three distinct mean pore sizes (94, 130, and 300 μm) by altering the freeze-drying technique used. It was evident that scaffolds with the largest mean pore sizes (300 μm) stimulated significantly higher cell proliferation, chondrogenic gene expression, cartilage-like matrix deposition, and resulting bulk compressive modulus after in vitro culture, relative to scaffolds with smaller mean pore sizes (94, 130 μm). Taken together, these findings demonstrate the importance of scaffold micro-architecture in the development of advanced tissue engineering strategies for articular cartilage defect repair.

Reliability of surface electromyography timing parameters in gait in cervical spondylotic myelopathy

The aims of this study were to validate a computerised method to detect muscle activity from surface electromyography (SEMG) signals in gait in patients with cervical spondylotic myelopathy (CSM), and to evaluate the test-retest reliability of the activation times designated by this method. SEMG signals were recorded from rectus femoris (RF), biceps femoris (BF), tibialis anterior (TA), and medial gastrocnemius (MG), during gait in 12 participants with CSM on two separate test days. Four computerised activity detection methods, based on the Teager-Kaiser Energy Operator (TKEO), were applied to a subset of signals and compared to visual interpretation of muscle activation. The most accurate method was then applied to all signals for evaluation of test-retest reliability. A detection method based on a combined slope and amplitude threshold showed the highest agreement (87.5%) with visual interpretation. With respect to reliability, the standard error of measurement (SEM) of the timing of RF, TA and MG between test days was 5.5% stride duration or less, while the SEM of BF was 9.4%. The timing parameters of RF, TA and MG designated by this method were considered sufficiently reliable for use in clinical practice, however the reliability of BF was questionable.

Enhanced bone healing using collagen-hydroxyapatite scaffold implantation in the treatment of a large multiloculated mandibular aneurysmal bone cyst in a thoroughbred filly.

An unmet need remains for a bone graft substitute material that is biocompatible, biodegradable and capable of promoting osteogenesis safely in vivo. The aim of this study was to investigate the use of a novel collagen–hydroxyapatite (CHA) bone graft substitute in the clinical treatment of a mandibular bone cyst in a young horse and to assess its potential to enhance repair of the affected bone. A 2 year‐old thoroughbred filly, presenting with a multilobulated aneurysmal bone cyst, was treated using the CHA scaffold. Post‐operative clinical follow‐up was carried out at 2 weeks and 3, 6 and 14 months. Cortical thickening in the affected area was observed from computed tomography (CT) examination as early as 3 months post‐surgery. At 14 months, reduced enlargement of the operated mandible was observed, with no fluid‐filled area. The expansile cavity was occupied by moderately dense mineralized tissue and fat and the compact bone was remodelled, with a clearer definition between cortex and medulla observed. This report demonstrates the promotion of enhanced bone repair following application of the CHA scaffold material in this craniomaxillofacial indication, and thus the potential of this material for translation to human applications. Copyright

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.

Effects of ageing, prolonged estrogen deficiency and zoledronate on bone tissue mineral distribution

The quantity and distribution of bone tissue mineral are key determinants of bone strength. Recent research revealed altered mineral distribution within sheep femora following estrogen deficiency. Rapid increases in bone remodeling occur at the onset of estrogen deficiency and abate over time. Therefore, altered tissue mineralization might be a transient characteristic of osteoporosis. Bisphosphonates reduce fracture incidence by 40-60% but increases in bone mineral density are insufficient to explain such changes. In this study the hypotheses that bone tissue mineralization is altered over prolonged estrogen depletion and bisphosphonate treatment were tested. Quantitative backscattered imaging (qBEI) was used to quantify bone mineral density distribution (BMDD) parameters (mean, FWHM) in trabeculae from the proximal femora of an ovariectomized sheep model that underwent estrogen deficiency for 31 months, an ovariectomized group administered with Zoledronic acid and age-matched controls. To assess the effects of normal ageing and prolonged estrogen deficiency, data were compared to BMDD data from sheep that were estrogen deficient for 12 months and age-matched controls. This study reports that normal ageing increases mean mineralization and mineral heterogeneity at a trabecular level. In contrast, prolonged estrogen deficiency leads to significantly decreased mean mineralization and further exacerbates increases in mineral heterogeneity. Interestingly, ZOL treatment of OVX sheep significantly reduced tissue mineral variability, both at a trabecular level and between femoral regions. Together, these findings indicate that ZOL treatment acts to reverse the increased mineral heterogeneity occurring during estrogen deficiency, which may contribute to its capacity to reduce osteoporotic fractures.