Development of a 3D-printed bioabsorbable composite scaffold with mechanical properties suitable for treating large, load-bearingarticular cartilage defects.

Extracellular matrix (ECM) biomaterials have shown promise for treating small artucular-joint defetcs. However, ECM-based biomaterials generally lack appropriate mechanical properties to support physiological loads and are prone to delamination in larger cartilage defects. To overcome these common mechanical limitations, a collagen hyaluronic-acid (CHyA) matrix, with proven regenerative potential, was reinforced with a bioabsorbable 3D-printed framework to support physiological loads. Polycaprolactone (PCL) was 3D-printed in two configurations, rectilinear and gyroid designs, that were extensively mechanically characterised. Both scaffold designs increased the compressive modulus of the CHyA matrices by three orders of magnitude, mimicking the physiological range (0.5-2.0 MPa) of healthy cartilage. The gyroid scaffold proved to be more flexible compared to the rectilinear scaffold, thus better contouring to the curvature of a femoral condyle. Additionally, PCL reinforcement of the CHyA matrix increased the tensile modulus and allowed for suture fixation of the scaffold to the subchondral bone, thus addressing the major challenge of biomaterial fixation to articular joint surfaces in shallow defects. In vitro evaluation confirmed successful infiltration of human mesenchymal stromal cells (MSCs) within the PCL-CHyA scaffolds, which resulted in increased production of sulphated glycosaminoglycans (sGAG/DNA; p = 0.0308) compared to non-reinforced CHyA matrices. Histological staining using alcian blue confirmed these results, while also indicating greater spatial distribution of sGAG throughout the PCL-CHyA scaffold. These findings have a great clinical importance as they provide evidence that reinforced PCL-CHyA scaffolds, with their increased chondroinductive potential and compatibility with joint fixation techniques, could be used to repair large-area chondral defects that currently lack effective treatment options.

Validation of a sub-epidermal moisture scanner for early detection of pressure ulcers in an ex vivo porcine model of localized oedema.

Pressure ulcers (PUs) remain a chronic health problem with severe impacts on healthcare systems. Early detection is crucial to providing effective interventions. However, detecting PUs currently relies on subjective tissue evaluations, such as visual skin assessment, precluding interventions prior to the development of visible tissue damage. There is an unmet need for solutions that can detect early tissue damage before visual and tactile signs occur. Assessments based on sub-epidermal moisture (SEM) measurements represent an opportunity for robust and objective early detection of PUs, preventing broken skin PUs in more high-risk patients at high-risk anatomical locations. While SEM assessment technology has been validated in computational, bench and tissue phantom models, validation in soft tissue was absent. In this study, we successfully validated the ability of a commercially available SEM assessment device to measure and detect sub-epidermal moisture changes in a novel ex vivo porcine soft tissue model of localised oedema. When controlled and incremental fluid volumes (Phosphate Buffer Solution) were injected into porcine soft tissues, statistically significant differences were found in SEM values between fluid-injected sites, representing an inflammatory oedematous condition, and healthy tissue control sites, as measured by the SEM device. The device provided reproducible readings by detecting localised oedema changes in soft tissues, reflecting the build-up of fluid as small as 1 ml into the underlying tissue. Spatial characterization experiments described the ability of the device technology to differentiate between healthy and oedematous tissue. Our findings validate the use of SEM assessment technology to measure and quantify localized oedema.

Development and in vitro investigation of a biodegradable mesh for the treatment of stress urinary incontinence.

INTRODUCTION AND HYPOTHESIS: The use of polypropylene (PP) mesh for stress urinary incontinence (SUI) surgery has declined because of safety concerns. The aim of this study is to evaluate a biodegradable polycaprolactone (PCL) mesh and a PCL composite mesh tissue engineered with human uterine fibroblasts (HUFs) for SUI surgery by comparing mechanical properties and in vitro biocompatibility to commercially available PP and porcine dermis (PD). METHODS: The mechanical properties of four scaffold materials were evaluated: PCL, PCL-collagen-hyaluronic acid composite, acellular porcine dermal collagen (PD) (Pelvicol™) and polypropylene (Gynecare TVT™ Exact®). HUFs were seeded on separate scaffolds. After 7 and 14 days scaffolds were assessed for metabolic activity and cell proliferation using Alamar Blue, Live/Dead and PicoGreen assays. Soluble collagen production was evaluated using a Sircol assay. RESULTS: PCL and the composite scaffold reached ultimate tensile strength (UTS) values closest to healthy pelvic floor tissue (PCL = 1.19 MPa; composite = 1.13 MPa; pelvic floor = 0.79 MPa; Lei et al. Int Urogynecol J Pelvic Floor Dysfunct. 18(6):603-7, 2007). Cells on PCL showed significantly greater cell viability than PP at day 7 (p < 0.0001). At D14 the composite scaffold showed significantly greater cell viability than PP (p = 0.0006). PCL was the best performing scaffold for soluble collagen production at day 14 (106.1 µg versus 13.04 µg for PP, p = 0.0173). CONCLUSIONS: We have designed a biodegradable PCL mesh and a composite mesh which demonstrate better biocompatibility than PP and mechanical properties closer to that of healthy pelvic floor tissue. This in vitro study provides promising evidence that these two implants should be evaluated in animal and human trials.

Tissue engineered extracellular matrices (ECMs) in urology: Evolution and future directions.

Autologous gastrointestinal tissue has remained the gold-standard reconstructive biomaterial in urology for >100 years. Mucus-secreting epithelium is associated with lifelong metabolic and neuromechanical complications when implanted into the urinary tract. Therefore, the availability of biocompatible tissue-engineered biomaterials such as extracellular matrix (ECM) scaffolds may provide an attractive alternative for urologists. ECMs are decellularised, biodegradable membranes that have shown promise for repairing defective urinary tract segments in vitro and in vivo by inducing a host-derived tissue remodelling response after implantation. In urology, porcine small intestinal submucosa (SIS) and porcine urinary bladder matrix (UBM) are commonly selected as ECMs for tissue regeneration. Both ECMs support ingrowth of native tissue and differentiation of multi-layered urothelial and smooth muscle cells layers while providing mechanical support in vivo. In their native acellular state, ECM scaffolds can repair small urinary tract defects. Larger urinary tract segments can be repaired when ECMs are manipulated by seeding them with various cell types prior to in vivo implantation. In the present review, we evaluate and summarise the clinical potential of tissue engineered ECMs in reconstructive urology with emphasis on their long-term outcomes in urological clinical trials.

An efficient, non-viral dendritic vector for gene delivery in tissue engineering.

Recent developments within the field of tissue engineering (TE) have shown that biomaterial scaffold systems can be augmented via the incorporation of gene therapeutics. The objective of this study was to assess the potential of the activated polyamidoamine dendrimer (dPAMAM) transfection reagent (SuperfectTM) as a gene delivery system to mesenchymal stem cells (MSCs) in both monolayer and 3D culture on collagen based scaffolds. dPAMAM-pDNA polyplexes at a mass ratio (M:R) 10:1 (dPAMAM : pDNA) (1 ug pDNA) were capable of facilitating prolonged reporter gene expression in monolayer MSCs which was superior to that facilitated using polyethylenimine (PEI)-pDNA polyplexes (2 ug pDNA). When dPAMAM-pDNA polyplexes (1 ug pDNA) were soak loaded onto a collagen-chondroitin sulphate (CS) scaffold prolonged transgene expression was facilitated which was higher than that obtained for a PEI-pDNA polyplex (2 ug pDNA) loaded scaffold. Transgene expression was dependent on the composite nature of the collagen scaffold with varying expression profiles obtained from a suite of collagen constructs including a collagen alone, collagen-CS, collagen-hydroxyapatite, collagen-nanohydroxyapatite and collagen-hyaluronic acid scaffold. Therefore, the dPAMAM vector described herein represents a biocompatible, effective gene delivery vector for TE applications which, via matching with a particular composite scaffold type, can be tailored for regeneration of various tissue defects.

Growth plate extracellular matrix-derived scaffolds for large bone defect healing.

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-1ß 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.

The effect of bisphosphonate treatment on the biochemical and cellular events during bone remodelling in response to microinjury stimulation.

Osteoporosis is one of the most prevalent bone diseases worldwide and is characterised by high levels of bone turnover, a marked loss in bone mass and accumulation of microdamage, which leads to an increased fracture incidence that places a huge burden on global health care systems. Bisphosphonates have been used to treat osteoporosis and have shown great success in conserving bone mass and reducing fracture incidence. In spite of the existing knowledge of the in vivo responses of bone to bisphosphonates, the cellular responses to these drugs have yet to be fully elucidated. In vitro model systems that allow the decoupling of complex highly integrated events, such as bone remodelling, provide a tool whereby these biological processes may be studied in a more simplified context. This study firstly utilised an in vitro model system of bone remodelling and comprising all three major cell types of the bone (osteocytes, osteoclasts and osteoblasts), which was representative of the bone's capacity to sense microdamage and subsequently initiate a basic multicellular unit response. Secondly, this system was used to study the effect of two commonly utilised aminobisphosphonate treatments for osteoporosis, alendronate and zoledronate. We demonstrated that microinjury to osteocyte networks being treated with bisphosphonates modulates receptor activator of nuclear factor kappa-B ligand and osteoprotegerin activity, and subsequently osteoclastogenesis. Furthermore, bisphosphonates increased the osteogenic potential following microinjury. Thus, we have shown for the first time that bisphosphonates act at all three stages of bone remodelling, from microinjury to osteoclastogenesis and ultimately osteogenesis.

Estrogen withdrawal from osteoblasts and osteocytes causes increased mineralization and apoptosis.

Recent studies have demonstrated increased bone mineral heterogeneity following estrogen withdrawal in vivo. Such changes likely contribute to fracture risk during post-menopausal osteoporosis since tissue mineralization is correlated with bone strength and stiffness. However, the cellular mechanisms responsible for increased mineral variability have not yet been distinguished. The objective of this study is to elucidate how alterations in mineral distribution are initiated during estrogen depletion. Specifically, we tested two separate hypotheses; (1) estrogen deficiency directly alters osteoblast mineralization and (2) estrogen deficiency increases bone cell apoptosis. Osteoblast-like cells (MC3T3-E1) and osteocyte-like cells (MLO-Y4) were pretreated with or without estrogen (17ß-estradiol) for 14 days. Estrogen deficiency was subsequently induced by either withdrawing estrogen from cells or blocking estrogen receptors using an estrogen antagonist, fulvestrant (ICI 182,780). Cell number (Hoechst DNA), alkaline phosphatase activity (p-NPP), mineralization (alizarin red) and apoptosis (Caspase 3/7) were evaluated. Whether estrogen withdrawal altered apoptosis rates in the presence of an apoptosis promoting agent (etoposide) was also determined. Interestingly, estrogen withdrawal from cells accustomed to estrogen exposure caused significantly increased osteoblast mineralization and osteocyte apoptosis compared with continued estrogen treatment. In contrast, blocking estrogen receptors with fulvestrant abrogated the mineralization induced by estrogen treatment. When apoptosis was induced using etoposide, cells undergoing estrogen withdrawal increased apoptosis compared to cells with continued estrogen treatment. Recognizing the underlying mechanisms regulating bone cell mineralization and apoptosis during estrogen deficiency and their consequences is necessary to further our knowledge of osteoporosis.

The osteogenic potential of the marine-derived multi-mineral formula aquamin is enhanced by the presence of vitamin D.

Bone degenerative diseases are on the increase globally and are often problematic to treat. This has led to a demand to identify supplements that aid bone growth and formation. Aquamin is a natural multi-mineral food supplement, derived from the red algae Lithothamnion species which contains calcium, magnesium and 72 other trace minerals. It has been previously reported to increase bone formation and mineralisation. This study aimed to investigate the 28 day in vitro osteogenic response of Aquamin supplemented with Vitamin D. The osteogenic potential of MC3T3-E1 osteoblast-like cells was analysed in standard osteogenic medium supplemented with Aquamin +/- Vitamin D3, and the controls consisted of osteogenic medium, +/- Vitamin D3. Proliferation of osteoblasts, metabolic activity and cell viability did not differ between Aquamin and the osteogenic control groups. Alkaline phosphatase (ALP) levels and mineralisation were increased by the supplementation of Aquamin, and the addition of Vitamin D3 increased mineralisation for all groups. The combination of Aquamin and Vitamin D3 yielded a significant increase in ALP and mineralisation over Aquamin alone and the standard osteogenic control +/- Vitamin D3. This study demonstrates that Aquamin aids osteogenesis, and that its osteogenic response can be enhanced by combining Aquamin with Vitamin D3.

Subchondral osteopenia and accelerated bone remodelling post-ovariectomy - a possible mechanism for subchondral microfractures in the aetiology of spontaneous osteonecrosis of the knee?

Osteopenia and subchondral microfractures are implicated in the aetiology of spontaneous osteonecrosis of the knee (SPONK). The ovine tibia shows significant alterations of the trabecular architecture within the subchondral bone of the medial tibial plateau post-ovariectomy (OVX), including reduced trabecular bone volume fraction. We hypothesise that accelerated subchondral bone resorption may also play a role in increasing microfracture risk at this site. Twenty-two sheep were examined in this study; 10 of the sheep underwent OVX, while the remainder (n = 13) were kept as controls (CON). Five fluorochrome dyes were administered intravenously at 12-week intervals via the jugular vein to both groups, to label sites of bone turnover. These animals were then killed at 12 months post-operatively. Bone turnover was significantly increased in the OVX group in both trabecular bone (2.024 vs. 1.047 no. mm(-2) ; P = 0.05) and within the subchondral bone plate (4.68 vs. 0.69 no. mm(-2) ; P < 0.001). In addition to the classically described turnover visible along trabecular surfaces, we also found visual evidence of intra-trabecular osteonal remodelling. In conclusion, this study shows significant alterations in bone turnover in both trabecular bone and within the subchondral bone plate at 1 year post-OVX. Remodelling of trabecular bone was due to both classically described hemi-osteonal and intra-trabecular osteonal remodelling. The presence of both localised osteopenia and accelerated bone remodelling within the medial tibial plateau provide a possible mechanism for subchondral microfractures in the aetiology of SPONK. Further utilisation of the OVX ewe may be useful for further study in this field.

Examination of osteoarthritis and subchondral bone alterations within the stifle joint of an ovariectomised ovine model.

The exact relationship between osteoporosis and osteoarthritis is still a matter for debate for many. The ovariectomised ewe is frequently used as a model for osteoporosis, resulting in significant alterations in bone morphometry and turnover in both trabecular and subchondral bone after 1 year. This study examines whether ovariectomy has any impact on development of osteoarthritis within the ovine stifle joint at the same time point. In addition, we investigate whether there are any significant correlations present between articular cartilage degeneration and alterations in microstructural parameters or turnover rates in the underlying bone. Twenty-two sheep were examined in this study; 10 of the sheep underwent ovariectomy and 12 were kept as controls. Five distinctive fluorochrome dyes were administered intravenously at 12-week intervals to both groups, to label sites of bone turnover. All animals were then sacrificed 12 months postoperatively. Although most specimens showed some evidence of osteoarthritis, no measurable difference between the two study groups was detected. Osteoarthritis was associated with a thinning of the subchondral plate, specifically the subchondral cortical bone; however, whereas previous studies have suggested a link between trabecular thinning and osteoarthritis, this was not confirmed. No correlation was found between osteoarthritis and bone turnover rates of either the subchondral trabecular bone or bone plate. In conclusion, despite the fact that ovariectomy results in marked morphological and structural changes in the ovine stifle joint at 1-year postoperatively, no evidence was found to suggest that it plays a direct role in the aetiology of osteoarthritis.

Influence of flow rate and scaffold pore size on cell behavior during mechanical stimulation in a flow perfusion bioreactor.

Mechanically stimulating cell-seeded scaffolds by flow-perfusion is one approach utilized for developing clinically applicable bone graft substitutes. A key challenge is determining the magnitude of stimuli to apply that enhances cell differentiation but minimizes cell detachment from the scaffold. In this study, we employed a combined computational modeling and experimental approach to examine how the scaffold mean pore size influences cell attachment morphology and subsequently impacts upon cell deformation and detachment when subjected to fluid-flow. Cell detachment from osteoblast-seeded collagen-GAG scaffolds was evaluated experimentally across a range of scaffold pore sizes subjected to different flow rates and exposure times in a perfusion bioreactor. Cell detachment was found to be proportional to flow rate and inversely proportional to pore size. Using this data, a theoretical model was derived that accurately predicted cell detachment as a function of mean shear stress, mean pore size, and time. Computational modeling of cell deformation in response to fluid flow showed the percentage of cells exceeding a critical threshold of deformation correlated with cell detachment experimentally and the majority of these cells were of a bridging morphology (cells stretched across pores). These findings will help researchers optimize the mean pore size of scaffolds and perfusion bioreactor operating conditions to manage cell detachment when mechanically simulating cells via flow perfusion.

Estrogen plus estrogen receptor antagonists alter mineral production by osteoblasts in vitro.

In early postmenopausal women, estrogen withdrawal is associated with increased bone turnover leading to bone loss and increased risk of fracture. Recent studies have suggested that the remaining bone tissue is significantly stronger, stiffer and has an increased tissue-level mineral content. Such changes may occur to compensate for bone loss or as a direct result of estrogen deficiency. To date many details of the physiology of osteoblastic cells during estrogen deficiency are vague. In this study we test the hypothesis that osteoblastic matrix mineralisation is altered at the onset of estrogen deficiency. In vitro cell culture experiments were carried out up to 28 days to compare the mineral production of MC3T3-E1 osteoblastic cells subject to estrogen deficiency (fulvestrant), enhanced estrogen supplementation (17-ß-estradiol) or a combination of both. Mineralisation was detected using von Kossa staining and was quantified with alizarin red absorbance readings. The expression of osteocalcin and osteopontin proteins, markers of osteoblast differentiation and mineralisation, was monitored using immunohistochemistry. Our results demonstrate that estrogen enhancement improves matrix mineralisation by MC3T3 cells in vitro. Furthermore this study found a significant reduction in the level of mineralisation when cells were treated with a combination of estrogen and fulvestrant. In an estrogen deficient environment mineralisation by osteoblastic cells was not altered. These findings suggest that altered tissue mineralisation following estrogen deficiency is not a direct result of estrogen deficiency on osteoblasts. Rather, we propose that altered tissue mineralisation may be a compensatory mechanism by bone to counter bone loss and reduced strength.

Subchondral trabecular structural changes in the proximal tibia in an ovine model of increased bone turnover.

Ovariectomized (OVX) sheep are now considered to be useful models for a variety of metabolic bone disorders. The specific aim of this study was to determine the effects of ovariectomy on the structural parameters and material density of the subchondral bone of the ovine tibial plateau as measured by microcomputed tomography (MicroCT). Twenty-three sheep were examined in this study; 10 of the sheep underwent ovariectomy (OVX), and the remainder (n=13) were kept as controls (CON). These animals were then sacrificed at 12 months post-operatively. Three-dimensional analyses were performed of osteochondral samples (15 mm deep) which were obtained from the medial tibial plateau using MicroCT. Bone volume fraction of the subchondral trabecular bone was reduced in the ovariectomized sheep as compared to control animals (0.439 vs. 0.483, P=0.038). Trabeculae were also significantly thinner in the OVX group (0.220 vs. 0.252 mm, P=0.010), with reduced connectivity density (7.947 vs. 11.524 mm(-3) , P=0.014). There was a trend towards lower numbers of individual trabeculae present in the OVX group as compared to controls, but this did not reach significance (2.817 vs. 3.288 mm(-1) , P=0.1). There was also increased trabecular separation in the OVX group, which again fell short of significance (0.426 vs. 0.387 mm, P=0.251). There was no difference in hydroxyapatite concentration (HA) between the two groups (929 vs. 932 mgHA cm(-3) , P=0.687). In conclusion, significant alterations of the trabecular architecture under the tibial plateau were observed following 12 months of oestrogen-deficiency in this ovine model. Despite these marked morphological and structural density differences, the material densities were equal in the two groups.

Site specific increase in heterogeneity of trabecular bone tissue mineral during oestrogen deficiency.

Although osteoporosis reduces overall bone mass causing bone fragility, recent studies report that the remaining bone tissue is significantly stiffer. Preliminary studies indicate that alterations in bone tissue mineral content might explain these changes, albeit that other studies report conflicting observations. The objective of this study is to quantify whether the distribution of bone tissue mineral is altered during oestrogen deficiency. Individual trabeculae were harvested from the proximal femur of 7 ovariectomised sheep (OVX), sacrificed 12 months post-surgery, and 5 age-matched controls. Mineral content (wt% Ca) was determined using a quantitative backscattered scanning electron microscopy imaging approach. Mineral heterogeneity within individual trabeculae was compared by calculating the full width at half maximum (FWHM) of mineral density distributions. Mean calcium content, the spatial distribution of mineral within trabeculae and the inter-trabecular variation between regions of proximal femora were also compared. Oestrogen deficiency increased mineral heterogeneity within individual trabeculae compared to healthy controls, as measured by FWHM (3.57 ± 0.68 vs. 3.17 ± 0.36 wt% Ca, p = 0.04). In particular mineral variability increased between superficial and deep regions of trabeculae of OVX animals (p = 0.04). Interestingly, mineralisation variability between greater and lesser trochanters (i.e. intertrochanteric fracture line) was increased in OVX compared to CON, as indicated by a greater % difference in the standard deviation of trabecular mineral content (77.11 ± 11.70 vs. 45.64 ± 23.70 %, p = 0.03). Such changes are undetectable by evaluating the mean mineral content of bone tissue, but may contribute to changes in bone mechanical strength following osteoporotic bone loss.

Biodegradable materials for surgical management of stress urinary incontinence: A narrative review.

Stress urinary incontinence (SUI) was managed with techniques such as colposuspension, autologous fascia sling and urethral bulking agents. The introduction of the mid-urethral polypropylene (PP) sling in the 1990s led to a significant and rapid global change in SUI surgery. The synthetic non-degradable PP sling had superior results to traditional SUI procedures but its use has now declined due to significant complications such as pain and mesh erosion. These complications are attributed to its poor biocompatibility and integration into vaginal tissues. The efficacy of PP was extrapolated from studies on abdominal wall repair and it is now clear that integration of implanted materials in the pelvic floor differs from the abdominal wall. With PP prohibited in some jurisdictions, female patients with SUI have few management options. In the present review we summarise recent advances in SUI surgery and evaluate potential alternatives to PP slings with a particular focus on degradable materials. Allograft and xenograft materials demonstrate good biocompatibility but have yielded suboptimal cure rates. Tissue engineered synthetic degradable materials outperform unmodified synthetic degradable materials in terms of biomechanics and cell support. Synthetic tissue engineered degradable materials show promising results from in vitro studies and future research should focus on animal and human trials in this field.

A new semi-orthotopic bone defect model for cell and biomaterial testing in regenerative medicine.

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.

Addition of hydroxyapatite improves stiffness, interconnectivity and osteogenic potential of a highly porous collagen-based scaffold for bone tissue regeneration.

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.

Anti-Ageing Protein ß-Klotho Rejuvenates Diabetic Stem Cells for Improved Gene-Activated Scaffold Based Wound Healing.

Skin wounds can lead to serious morbidity complications in diabetic patients due to the reduced healing potential of autologous stem cells. One reason for the low functional potency of stem cells from diabetic patients (diabetic stem cells) is attributed to their senescent-like nature. Here, we investigated if an anti-ageing protein, ß-klotho, could be used to rejuvenate diabetic stem cells and to promote pro-angiogenic gene-activated scaffold (GAS)-induced functional response for wound healing applications. Human stem cells derived from the adipose tissue (adipose-derived stem cells (ADSCs)) of normal and diabetic (type 2) donors were used for the study. We report that the ß-klotho priming facilitated inflammatory signal pruning by reducing interleukin-8 release by more than half while concurrently doubling the release of monocyte chemoattractant protein-1. Additionally, ß-klotho priming enhanced the pro-angiogenic response of diabetic ADSCs on GAS by dampening the release of anti-angiogenic factors (i.e., pigment epithelium-derived factor, tissue inhibitor of metalloproteinase-1 and thrombospondin-1) while simultaneously supporting the expression of pro-angiogenic factors (i.e., Vascular Endothelial Growth Factor (VEGF), angiopoietin-2 and angiogenin). Finally, we show that ß-klotho pre-treatment expedites the cellular expression of matrix proteins such as collagen IV and collagen VI, which are implicated in tissue maturation. Taken together, our study provides evidence that the synergistic effect of the pro-angiogenic GAS and ß-klotho activation effectively accelerates the functional development of diabetic ADSCs for wound healing applications.

Biomaterial-based endochondral bone regeneration: a shift from traditional tissue engineering paradigms to developmentally inspired strategies.

There is an urgent, clinical need for an alternative to the use of autologous grafts for the ever increasing number of bone grafting procedures performed annually. Herein, we describe a developmentally inspired approach to bone tissue engineering, which focuses on leveraging biomaterials as platforms for recapitulating the process of endochondral ossification. To begin, we describe the traditional biomaterial-based approaches to tissue engineering that have been investigated as methods to promote in vivo bone regeneration, including the use of three-dimensional biomimetic scaffolds, the delivery of growth factors and recombinant proteins, and the in vitro engineering of mineralized bone-like tissue. Thereafter, we suggest that some of the hurdles encountered by these traditional tissue engineering approaches may be circumvented by modulating the endochondral route to bone repair and, to that end, we assess various biomaterials that can be used in combination with cells and signaling factors to engineer hypertrophic cartilaginous grafts capable of promoting endochondral bone formation. Finally, we examine the emerging trends in biomaterial-based approaches to endochondral bone regeneration, such as the engineering of anatomically shaped templates for bone and osteochondral tissue engineering, the fabrication of mechanically reinforced constructs using emerging three-dimensional bioprinting techniques, and the generation of gene-activated scaffolds, which may accelerate the field towards its ultimate goal of clinically successful bone organ regeneration.