The chondro-osseous continuum: is it possible to unlock the potential assigned within?

Endochondral ossification (EO), by which long bones of the axial skeleton form, is a tightly regulated process involving chondrocyte maturation with successive stages of proliferation, maturation, and hypertrophy, accompanied by cartilage matrix synthesis, calcification, and angiogenesis, followed by osteoblast-mediated ossification. This developmental sequence reappears during fracture repair and in osteoarthritic etiopathology. These similarities suggest that EO, and the cells involved, are of great clinical importance for bone regeneration as it could provide novel targeted approaches to increase specific signaling to promote fracture healing, and if regulated appropriately in the treatment of osteoarthritis. The long-held accepted dogma states that hypertrophic chondrocytes are terminally differentiated and will eventually undergo apoptosis. In this mini review, we will explore recent evidence from experiments that revisit the idea that hypertrophic chondrocytes have pluripotent capacity and may instead transdifferentiate into a specific sub-population of osteoblast cells. There are multiple lines of evidence, including our own, showing that local, selective alterations in cartilage extracellular matrix (ECM) remodeling also indelibly alter bone quality. This would be consistent with the hypothesis that osteoblast behavior in long bones is regulated by a combination of their lineage origins and the epigenetic effects of chondrocyte-derived ECM which they encounter during their recruitment. Further exploration of these processes could help to unlock potential novel targets for bone repair and regeneration and in the treatment of osteoarthritis

Dysfunctional stem and progenitor cells impair fracture healing with age

Successful fracture healing requires the simultaneous regeneration of both the bone and vasculature; mesenchymal stem cells (MSCs) are directed to replace the bone tissue, while endothelial progenitor cells (EPCs) form the new vasculature that supplies blood to the fracture site. In the elderly, the healing process is slowed, partly due to decreased regenerative function of these stem and progenitor cells. MSCs from older individuals are impaired with regard to cell number, proliferative capacity, ability to migrate, and osteochondrogenic differentiation potential. The proliferation, migration and function of EPCs are also compromised with advanced age. Although the reasons for cellular dysfunction with age are complex and multidimensional, reduced expression of growth factors, accumulation of oxidative damage from reactive oxygen species, and altered signaling of the Sirtuin-1 pathway are contributing factors to aging at the cellular level of both MSCs and EPCs. Because of these geriatric-specific issues, effective treatment for fracture repair may require new therapeutic techniques to restore cellular function. Some suggested directions for potential treatments include cellular therapies, pharmacological agents, treatments targeting age-related molecular mechanisms, and physical therapeutics. Advanced age is the primary risk factor for a fracture, due to the low bone mass and inferior bone quality associated with aging; a better understanding of the dysfunctional behavior of the aging cell will provide a foundation for new treatments to decrease healing time and reduce the development of complications during the extended recovery from fracture healing in the elderly

Healing of Osteochondral Defects via Endochondral Ossification in an Ovine Model

© The Author(s) 2017. Objective: The objective of this study was to describe the mechanism of healing of osteochondral defects of the distal femur in the sheep, a commonly used translational model. Information on the healing mechanism be useful to inform the design of tissue engineering devices for joint surface defect repair. Design: A retrospective study was conducted examining 7-mm diameter osteochondral defects made in the distal medial femoral condyle of 40 adult female sheep, comprising control animals from 3 separate structures. The healing of the defects was studied at post mortem at up to 26 weeks. Results: Osteochondral defects of the distal femur of the sheep heal through endochondral ossification as evidenced by chondrocyte hypertrophy and type X collagen expression. Neocartilage is first formed adjacent to damaged cartilage and then streams over the damaged underlying bone before filling the defect from the base upward. No intramembranous ossification or isolated mesenchymal stem cell aggregates were detected in the healing tissue. No osseous hypertrophy was detected in the defects. Conclusions: Osteochondral defects of the medial femoral condyle of the sheep heal via endochondral ossification, with neocartilage first appearing adjacent to damaged cartilage. Unlike the mechanism of healing in fracture repair, neocartilage is eventually formed directly onto damaged bone. There was most variability between animals between 8 and 12 weeks postsurgery. These results should be considered when designing devices to promote defect healing

Understanding cell source and extracellular matrix contributions to cartilage and bone repair for regenerative medicine applications

Human skeletal elements are grossly divided into three main tissue categories: bone, cartilage and muscle. While skeletal muscle is closely associated and interacts with the bony element, this thesis focuses specifically on the repair mechanisms involved in bone and cartilage and how to better mediate these mechanisms from a regenerative medicine perspective; muscle will not be treated hereafter and any reference to skeletal tissue refers either to bone or cartilage. To begin, I will first define key concepts in skeletal tissue repair that give background to the regenerative strategies chosen during this thesis. Following this, I will introduce the concept of tissue engineering and the parameters that are necessary to take into account when preparing a living tissue graft. After this brief introduction, I will present the experimental work performed during this thesis in which the specific strategies employed towards skeletal tissue engineering are presented. Finally, I conclude with a summary of accomplishments and suggest further work that could be performed to help advance the presented topics towards a translational technology

A perfusion culture system for assessing bone marrow stromal cell differentiation on PLGA scaffolds for bone repair

Biomaterials development for bone repair is currently hindered by the lack of physiologically relevant in vitro testing systems. Here we describe the novel use of a bi-directional perfusion bioreactor to support the long term culture of human bone marrow stromal cells (BMSCs) differentiated on polylactic co-glycolic acid (PLGA). Primary human BMSCs were seeded onto porous PLGA scaffolds and cultured in static vs. perfusion culture conditions for 21 days in osteogenic vs. control media. PLGA scaffolds were osteoconductive, supporting a mature osteogenic phenotype as shown by the upregulation of Runx2 and the early osteocyte marker E11. Perfusion culture enhanced the expression of osteogenic genes Osteocalcin and Osteopontin. Extracellular matrix deposition and mineralisation were spatially regulated within PLGA scaffolds in a donor dependant manner. This, together with the observed upregulation of Collagen type X suggested an environment permissive for the study of differentiation pathways associated with both intramembranous and endochondral ossification routes of bone healing. This culture system offers a platform to assess BMSC behavior on candidate biomaterials under physiologically relevant conditions. Use of this system may improve our understanding of the environmental cues orchestrating BMSC differentiation and enable fine tuning of biomaterial design as we develop tissue-engineered strategies for bone regeneration

Resting and injury-induced inflamed periosteum contain multiple macrophage subsets that are located at sites of bone growth and regeneration

Better understanding of bone growth and regeneration mechanisms within periosteal tissues will improve understanding of bone physiology and pathology. Macrophage contributions to bone biology and repair have been established but specific investigation of periosteal macrophages has not been undertaken. We used an immunohistochemistry approach to characterize macrophages in growing murine bone and within activated periosteum induced in a mouse model of bone injury. Osteal tissue macrophages (osteomacs) and resident macrophages were distributed throughout resting periosteum. In tissues collected from 4-week-old mice, osteomacs were observed intimately associated with sites of periosteal diaphyseal and metaphyseal bone dynamics associated with normal growth. This included F4/80(+)Mac-2(-/low) osteomac association with extended tracks of bone formation (modeling) on diphyseal periosteal surfaces. Although this recapitulated endosteal osteomac characteristics, there was subtle variance in the morphology and spatial organization of periosteal modeling-associated osteomacs, which likely reflects.the greater structural complexity of periosteum. Osteomacs, resident macrophages and inflammatory macrophages (F4/80(+)Mac-2(hi) were associated with the complex bone dynamics occurring within the periosteum at the metaphyseal corticalization zone. These three macrophage subsets were also present within activated native periosteum after bone injury across a 9-day time course that spanned the inflammatory through remodeling bone healing phases. This included osteomac association with foci of endochondral ossification within the activated native periosteum. These observations confirm that osteomacs are key components of both osteal tissues, in spite of salient differences between endosteal and periosteal structure and that multiple macrophage subsets are involved in periosteal bone dynamics

Healing of Osteochondral Defects via Endochondral Ossification in an Ovine Model.

OBJECTIVE: The objective of this study was to describe the mechanism of healing of osteochondral defects of the distal femur in the sheep, a commonly used translational model. Information on the healing mechanism be useful to inform the design of tissue engineering devices for joint surface defect repair. DESIGN: A retrospective study was conducted examining 7-mm diameter osteochondral defects made in the distal medial femoral condyle of 40 adult female sheep, comprising control animals from 3 separate structures. The healing of the defects was studied at post mortem at up to 26 weeks. RESULTS: Osteochondral defects of the distal femur of the sheep heal through endochondral ossification as evidenced by chondrocyte hypertrophy and type X collagen expression. Neocartilage is first formed adjacent to damaged cartilage and then streams over the damaged underlying bone before filling the defect from the base upward. No intramembranous ossification or isolated mesenchymal stem cell aggregates were detected in the healing tissue. No osseous hypertrophy was detected in the defects. CONCLUSIONS: Osteochondral defects of the medial femoral condyle of the sheep heal via endochondral ossification, with neocartilage first appearing adjacent to damaged cartilage. Unlike the mechanism of healing in fracture repair, neocartilage is eventually formed directly onto damaged bone. There was most variability between animals between 8 and 12 weeks postsurgery. These results should be considered when designing devices to promote defect healing

Biomimetic strategies for fracture repair: engineering the cell microenvironment for directed tissue formation

Complications resulting from impaired fracture healing have major clinical implications on fracture management strategies. Novel concepts taken from developmental biology have driven research strategies towards the elaboration of regenerative approaches that can truly harness the complex cellular events involved in tissue formation and repair. Advances in polymer technology and a better understanding of naturally derived scaffolds have given rise to novel biomaterials with an increasing ability to recapitulate native tissue environments. This coupled with advances in the understanding of stem cell biology and technology has opened new avenues for regenerative strategies with true clinical translatability. These advances have provided the impetus to develop alternative approaches to enhance the fracture repair process. We provide an update on these advances, with a focus on the development of novel biomimetic approaches for bone regeneration and their translational potential