C Roberts

Evaluation of skeletal tissue repair, Part 1: Assessment of novel growth-factor-releasing hydrogels in an ex vivo chick femur defect model

Current clinical treatments for skeletal conditions resulting in large-scale bone loss include autograft or allograft, both of which have limited effectiveness. In seeking to address bone regeneration, several tissue engineering strategies have come to the fore, including the development of growth factor releasing technologies and appropriate animal models to evaluate repair. Ex vivo models represent a promising alternative to simple in vitro systems or complex, ethically challenging in vivo models. We have developed an ex vivo culture system of whole embryonic chick femora, adapted in this study as a critical size defect model to investigate the effects of novel bone extracellular matrix (bECM) hydrogel scaffolds containing spatio-temporal growth factor-releasing microparticles and skeletal stem cells on bone regeneration, to develop a viable alternative treatment for skeletal degeneration. Alginate/bECM hydrogels combined with poly (d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PDLLGA) microparticles releasing VEGF, TGF-β3 or BMP-2 were placed, with human adult Stro-1+ bone marrow stromal cells, into 2mm central segmental defects in embryonic chick femurs. Alginate/bECM hydrogels loaded with HSA/VEGF or HSA/TGF-β3 demonstrated a cartilage-like phenotype, with minimal collagen I deposition, comparable to HSA-only control hydrogels. The addition of BMP-2 releasing microparticles resulted in enhanced structured bone matrix formation, evidenced by increased Sirius red-stained matrix and collagen expression within hydrogels. This study demonstrates delivery of bioactive growth factors from a novel alginate/bECM hydrogel to augment skeletal tissue formation and the use of an organotypic chick femur defect culture system as a high-throughput test model for scaffold/cell/growth factor therapies for regenerative medicine.

Evaluation of skeletal tissue repair, Part 2: Enhancement of skeletal tissue repair through dual-growth-factor-releasing hydrogels within an ex vivo chick femur defect model

There is an unmet need for improved, effective tissue engineering strategies to replace or repair bone damaged through disease or injury. Recent research has focused on developing biomaterial scaffolds capable of spatially and temporally releasing combinations of bioactive growth factors, rather than individual molecules, to recapitulate repair pathways present in vivo. We have developed an ex vivo embryonic chick femur critical size defect model and applied the model in the study of novel extracellular matrix (ECM) hydrogel scaffolds containing spatio-temporal combinatorial growth factor-releasing microparticles and skeletal stem cells for bone regeneration. Alginate/bovine bone ECM (bECM) hydrogels combined with poly(d,l-lactic-co-glycolic acid) (PDLLGA)/triblock copolymer (10-30% PDLLGA-PEG-PLDLGA) microparticles releasing dual combinations of vascular endothelial growth factor (VEGF), chondrogenic transforming growth factor beta 3 (TGF-β3) and the bone morphogenetic protein BMP2, with human adult Stro-1+bone marrow stromal cells (HBMSCs), were placed into 2mm central segmental defects in embryonic day 11 chick femurs and organotypically cultured. Hydrogels loaded with VEGF combinations induced host cell migration and type I collagen deposition. Combinations of TGF-β3/BMP2, particularly with Stro-1+HBMSCs, induced significant formation of structured bone matrix, evidenced by increased Sirius red-stained matrix together with collagen expression demonstrating birefringent alignment within hydrogels. This study demonstrates the successful use of the chick femur organotypic culture system as a high-throughput test model for scaffold/cell/growth factor therapies in regenerative medicine. Temporal release of dual growth factors, combined with enriched Stro-1+HBMSCs, improved the formation of a highly structured bone matrix compared to single release modalities. These studies highlight the potential of a unique alginate/bECM hydrogel dual growth factor release platform for bone repair.

In vivo assessment of bone regeneration in alginate/bone ECM hydrogels with incorporated skeletal stem cells and single growth factors

The current study has investigated the use of decellularised, demineralised bone extracellular matrix (ECM) hydrogel constructs for in vivo tissue mineralisation and bone formation. Stro-1-enriched human bone marrow stromal cells were incorporated together with select growth factors including VEGF, TGF-β3, BMP-2, PTHrP and VitD3, to augment bone formation, and mixed with alginate for structural support. Growth factors were delivered through fast (non-osteogenic factors) and slow (osteogenic factors) release PLGA microparticles. Constructs of 5 mm length were implanted in vivo for 28 days within mice. Dense tissue assessed by micro-CT correlated with histologically assessed mineralised bone formation in all constructs. Exogenous growth factor addition did not enhance bone formation further compared to alginate/bone ECM (ALG/ECM) hydrogels alone. UV irradiation reduced bone formation through degradation of intrinsic growth factors within the bone ECM component and possibly also ECM cross-linking. BMP-2 and VitD3 rescued osteogenic induction. ALG/ECM hydrogels appeared highly osteoinductive and delivery of angiogenic or chondrogenic growth factors led to altered bone formation. All constructs demonstrated extensive host tissue invasion and vascularisation aiding integration and implant longevity. The proposed hydrogel system functioned without the need for growth factor incorporation or an exogenous inducible cell source. Optimal growth factor concentrations and spatiotemporal release profiles require further assessment, as the bone ECM component may suffer batch variability between donor materials. In summary, ALG/ECM hydrogels provide a versatile biomaterial scaffold for utilisation within regenerative medicine which may be tailored, ultimately, to form the tissue of choice through incorporation of select growth factors.

Effects of hypothyroidism on the structure and mechanical properties of bone in the ovine fetus

Thyroid hormones are important for normal bone growth and development in postnatal life. However, little is known about the role of thyroid hormones in the control of bone development in the fetus. Using computed tomography and mechanical testing, the structure and strength of metatarsal bones were measured in sheep fetuses in which thyroid hormone levels were altered by thyroidectomy or adrenalectomy. In intact fetuses, plasma concentrations of total calcium and the degradation products of C-terminal telopeptides of type I collagen increased between 100 and 144 days of gestation (term 145±2 days), in association with various indices of bone growth and development. Thyroid hormone deficiency induced by thyroidectomy at 105-110 days of gestation caused growth retardation of the fetus and significant changes in metatarsal bone structure and strength when analyzed at both 130 and 144 days of gestation. In hypothyroid fetuses, trabecular bone was stronger with thicker, more closely spaced trabeculae, despite lower bone mineral density. Plasma osteocalcin was reduced by fetal thyroidectomy. Removal of the fetal adrenal gland at 115-120 days of gestation, and prevention of the prepartum rises in cortisol and triiodothyronine, had no effect on bodyweight, limb lengths, metatarsal bone structure or strength, or circulating markers of bone metabolism in the fetuses studied near term. This study demonstrates that hypothyroidism in utero has significant effects on the structure and strength of bone, with different consequences for cortical and trabecular bone.

Effect of Vitamin D deficiency during pregnancy on offspring bone structure, composition and quality in later life

During foetal development, calcium requirements are met as a consequence of maternal adaptations independent of vitamin D status. In contrast, after birth, dependency on vitamin D appears necessary for calcium metabolism and skeletal health. We used a rodent model (Sprague-Dawley rats), to determine if maternal vitamin D deficiency during pregnancy had a deleterious effect on bone structure at birth. Vitamin D deplete females were maintained under deplete conditions until birth of the pups, whereupon all dams were fed a vitamin D replete diet. Offspring were harvested at birth, and 140 days of age. Bones were analyzed using micro-computed tomography and strength tested to study differences in bone structure, density and strength and subjected to elemental analysis using plasma mass spectrometry to determine strontium, barium and calcium contents. Offspring from deplete mothers displayed altered trabecular parameters in the femur at birth and 140 days of age. In addition, at 140 days of age there was evidence of premature mineralization of the secondary ossification centre of the femoral head. Elemental analysis showed increased strontium uptake in the femur of the developmentally vitamin D-deficient offspring. Vitamin D depletion during development in the offspring may have a long-lasting effect, despite repletion of vitamin D from birth. This may have consequences for human health given the low vitamin D levels seen during pregnancy and current lifestyle of sun avoidance due to the risk of skin cancer.