Characterisation and evaluation of the regenerative capacity of Stro-4+ enriched bone marrow mesenchymal stromal cells using bovine extracellular matrix hydrogel and a novel biocompatible melt electro-written medical-grade polycaprolactone scaffold

Many skeletal tissue regenerative strategies centre around the multifunctional properties of bone marrow derived stromal cells (BMSC) or mesenchymal stem/stromal cells (MSC)/bone marrow derived skeletal stem cells (SSC). Specific identification of these particular stem cells has been inconclusive. However, enriching these heterogeneous bone marrow cell populations with characterised skeletal progenitor markers has been a contributing factor in successful skeletal bone regeneration and repair strategies. In the current studies we have isolated, characterised and enriched ovine bone marrow mesenchymal stromal cells (oBMSCs) using a specific antibody, Stro-4, examined their multipotential differentiation capacity and, in translational studies combined Stro-4+ oBMSCs with a bovine extracellular matrix (bECM) hydrogel and a biocompatible melt electro-written medical-grade polycaprolactone scaffold, and tested their bone regenerative capacity in a small in vivo, highly vascularised, chick chorioallantoic membrane (CAM) model and a preclinical, critical-sized ovine segmental tibial defect model.Proliferation rates and CFU-F formation were similar between unselected and Stro-4+ oBMSCs. Col1A1, Col2A1, mSOX-9, PPARG gene expression were upregulated in respective osteogenic, chondrogenic and adipogenic culture conditions compared to basal conditions with no significant difference between Stro-4+ and unselected oBMSCs. In contrast, proteoglycan expression, alkaline phosphatase activity and adipogenesis were significantly upregulated in the Stro-4+ cells. Furthermore, with extended cultures, the oBMSCs had a predisposition to maintain a strong chondrogenic phenotype. In the CAM model Stro-4+ oBMSCs/bECM hydrogel was able to induce bone formation at a femur fracture site compared to bECM hydrogel and control blank defect alone. Translational studies in a critical-sized ovine tibial defect showed autograft samples contained significantly more bone, (4250.63 mm3, SD = 1485.57) than blank (1045.29 mm3, SD = 219.68) ECM-hydrogel (1152.58 mm3, SD = 191.95) and Stro-4+/ECM-hydrogel (1127.95 mm3, SD = 166.44) groups.Stro-4+ oBMSCs demonstrated a potential to aid bone repair in vitro and in a small in vivo bone defect model using select scaffolds. However, critically, translation to a large related preclinical model demonstrated the complexities of bringing small scale reported stem-cell material therapies to a clinically relevant model and thus facilitate progression to the clinic

Growth factors for skeletal reconstruction and fracture repair

The demographic challenges of an ageing population have emphasized the need for processes to augment and repair skeletal tissue loss as a consequence of trauma and/or degeneration. A number of bone growth factors have been shown to be expressed during the course of fracture healing, suggesting a potential role in bone and cartilage formation, and in fracture repair. This review focuses on a select number of these growth factors currently under preclinical and clinical evaluation for skeletal regeneration and fracture repair. The limitations in the use of these skeletal factors to augment bone growth, thus improving quality-of-life and reducing the significant social and economic costs associated with skeletal trauma/loss are also considered

In vitro and in vivo methods to determine the interactions of osteogenic cells with biomaterials

To assess new biomaterials for possible use as bone graft substitutes, a number of techniques allow interactions with osteoblastic cells to be studied, with respect to effects on proliferation and differentiation of osteoprogenitors. In vitro models include the use of bone explant cultures, fetal rat calvarial-derived osteoblast cells, primary stromal populations, transformed and non-transformed cell lines and immortalized osteoblast cell lines. However, these assessments are limited by the extent of osteogenic differentiation and bone formation that can be observed in vitro, species differences and phenotypic drift of cells cultured in vitro. The use of in vivo experimental systems such as the segmental/calvarial bone defect model, the subcutaneous implant model and the diffusion chamber implantation model circumvent some of these issues and, in the appropriate model, provide data on efficacy, biocompatibility and osteointegration of a biomaterial. The combination of in vitro and in vivo approaches together with the development of new cell labeling techniques, in particular the ability to genetically mark and select specific human bone cell populations provides new avenues for their potential evaluation in combination with appropriate biomaterials for clinical use. These in vitro and in vivo techniques are reviewed and those recently developed for assessment of human osteogenic cells should be applicable to many other cell systems where knowledge of specific human tissue or cell interactions with biomaterials is required

Osteogenesis and angiogenesis: the potential for engineering bone

The repair of large bone defects remains a major clinical orthopaedic challenge. Bone is a highly vascularised tissue reliant on the close spatial and temporal connection between blood vessels and bone cells to maintain skeletal integrity. Angiogenesis thus plays a pivotal role in skeletal development and bone fracture repair. Current procedures to repair bone defects and to provide structural and mechanical support include the use of grafts (autologous, allogeneic) or implants (polymeric or metallic). These approaches face significant limitations due to insufficient supply, potential disease transmission, rejection, cost and the inability to integrate with the surrounding host tissue. The engineering of bone tissue offers new therapeutic strategies to aid musculoskeletal healing. Various scaffold constructs have been employed in the development of tissue-engineered bone; however, an active blood vessel network is an essential pre-requisite for these to survive and integrate with existing host tissue. Combination therapies of stem cells and polymeric growth factor release scaffolds tailored to promote angiogenesis and osteogenesis are under evaluation and development actively to stimulate bone regeneration. An understanding of the cellular and molecular interactions of blood vessels and bone cells will enhance and aid the successful development of future vascularised bone scaffold constructs, enabling survival and integration of bioengineered bone with the host tissue. The role of angiogenic and osteogenic factors in the adaptive response and interaction of osteoblasts and endothelial cells during the multi step process of bone development and repair will be highlighted in this review, with consideration of how some of these key mechanisms can be combined with new developments in tissue engineering to enable repair and growth of skeletal fractures. Elucidation of the processes of angiogenesis, osteogenesis and tissue engineering strategies offer exciting future therapeutic opportunities for skeletal repair and regeneration in orthopaedics

Delivery systems for bone growth factors - the new players in skeletal regeneration

Given the challenge of an increasing elderly population, the ability to repair and regenerate traumatised or lost tissue is a major clinical and socio-economic need. Pivotal in this process will be the ability to deliver appropriate growth factors in the repair cascade in a temporal and tightly regulated sequence using appropriately designed matrices and release technologies within a tissue engineering strategy. This review outlines the current concepts and challenges in growth factor delivery for skeletal regeneration and the potential of novel delivery matrices and biotechnologies to influence the healthcare of an increasing ageing population

Assessing the potential of colony morphology for dissecting the CFU-F population from human bone marrow stromal cells

Mesenchymal stem cells (MSCs) provide an ideal cell source for bone tissue engineering strategies. However, bone marrow stromal cell (BMSC) populations that contain MSCs are highly heterogeneous expressing a wide variety of proliferative and differentiation potentials. Current MSC isolation methods employing magnetic-activated and fluorescent-activated cell sorting can be expensive and time consuming and, in the absence of specific MSC markers, fail to generate homogeneous populations. We have investigated the potential of various colony morphology descriptors to provide correlations with cell growth potential. Density-independent colony forming unit-fibroblastic (CFU-F) capacity is a MSC prerequisite and resultant colonies display an array of shapes and sizes that might be representative of cell function. Parent colonies were initially categorised according to their diameter and cell density and grouped before passage for the subsequent assessment of progeny colonies. Whereas significant morphological differences between distinct parent populations indicated a correlation with immunophenotype, enhanced CFU-F capacity was not observed when individual colonies were isolated according to these morphological parameters. Colony circularity, an alternative morphological measure, displayed a strong correlation with subsequent cell growth potential. The current study indicates the potential of morphological descriptors for predicting cell growth rate and suggests new directions for research into dissection of human BMSC CFU-F populations

Skeletal progenitor cells and ageing human populations

1. Stem and progenitor cells present within bone marrow give rise to colony forming units-fibroblastic (CFU-F) which can differentiate into fibroblastic, osteogenic, myogenic, adipogenic and reticular cells. The decrease in skeletal bone formation and rate of fracture repair observed with ageing and in osteoporosis has been suggested to be due to a decrease in numbers of these progenitors, but human studies are limited. 2. We have tested the potential to form CFU-F in a total of 99 patients undergoing corrective surgery (16 controls, 14-48 years of age) or hip arthroplasty for osteoarthritis (57 patients, 28-87 years of age) or osteoporosis (26 patients, 69-97 years of age). Total colony number, alkaline phosphatase-positive colony number and colony size were determined. 3. No decrease in colony forming efficiency under the culture conditions used was observed in all populations examined irrespective of age, disease or gender, as determined by the lack of correlation between colony formation and age. 3. Examination of colony sizes showed a significant reduction in colony size with age in osteoarthritis and in control populations indicating a change in cellular proliferative potential with age. 4. Examination of number and percentage of alkaline phosphatase-positive CFU-F showed a significant decrease in osteoporotic patients compared with controls and osteoarthritis patients, indicating altered differentiation potential. 5. These results suggest that the reduction in bone mass with ageing may be due to reduction of the proliferative capacity of progenitor cells or their responsiveness to biological factors leading to alteration in subsequent differentiation. The maintenance of CFU-F number and alkaline phosphatase activity in these osteoarthritis patients may, in part, explain the inverse relationship observed for the preservation of bone mass between generalized osteoarthritis and primary osteoporosi