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

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

IGF-I increases bFGF-induced mitogenesis and upregulates FGFR-1 in rabbit vascular smooth muscle cells

Insulin-like growth factor-I (IGF-I) and basic fibroblast growth factor (bFGF) have both been implicated in the abnormal proliferation of vascular smooth muscle cells (VSMC) that occurs after injury to the arterial wall in vivo. We have investigated the effects of these growth factors on proliferation of rabbit aortic smooth muscle cells (RASMC) in vitro. IGF-I, in contrast to bFGF, is a weak mitogen for RASMC. However, when IGF-I (10 ng/ml) was added in combination with bFGF for 24 h, the effect of the two growth factors on DNA synthesis was synergistic at all concentrations tested (P &gt; 0.001 compared with summed values of bFGF alone plus IGF-I alone), and this synergy was also observed at the level of RASMC proliferation (P &lt; 0.001). Time-course experiments indicated that although bFGF was able to stimulate DNA synthesis after 16 h, activity peaked at 24 h, and a synergistic response with IGF-I was not observed before 24 h. Northern blot analysis demonstrated that IGF-I (10 ng/ml) could selectively upregulate fibroblast growth factor receptor-1 (FGFR-1) mRNA 4.0 +/- 0.24-fold (P &lt; 0.001) without a significant effect on FGFR-2, and this induction in FGFR-1 mRNA occurs in a time- and dose-dependent manner. In addition, IGF-I increases FGFR-1 protein levels in RASMC 2.7 +/- 0.12-fold (P &lt; 0.01), as demonstrated by Western blotting, and this upregulation occurs before the peak in DNA synthesis. These results suggest that IGF-I may be capable of increasing the responsiveness of VSMC to bFGF through modulation of FGFR-1. <br/

Development of in vivo muCT evaluation of neovascularisation in tissue engineered bone constructs

Due to an increasing aging population the need for innovative approaches to aid skeletal repair and reconstruction is a significant socio-economic increasing problem. The emerging discipline of tissue engineering has sort to augment the growth and repair of bone loss particularly in areas of trauma, degeneration and revision surgery. However, the initiation and development of a fully functional vascular network are critical for bioengineered bone to repair large osseous defects, whether the material is osteosynthetic (poly (d,l)-lactic acid, PLA) or natural bone allograft. Quantification and three-dimensional visualization of new vessel networks remain a problem in bone tissue engineering constructs. A novel technique utilising a radio-opaque dye and micro-computed tomography (muCT) has been developed and applied to study angiogenesis in an impaction bone graft model. Tissue-engineered constructs combining human bone marrow stromal cells (HBMSC) with natural allograft and synthetic grafts (PLA) were impacted and implanted into the subcutis of MF-1 nu/nu mice for a period of 28 days. Microfil consisting of radio-opaque polymer was perfused through the mice and scanned using a Bench Top CT system for micro-computed tomography. Analysis of three-dimensional muCT reconstructions demonstrated an increase in vessel volume and vessel number in the impacted scaffolds/HBMC compared to scaffolds alone. Vessel volume: allograft/HBMSC=0.57 mm(3)+/-0.19; allograft=0.04 mm(3)+/-0.04; PLA/HBMSC=1.19 mm(3)+/-0.31; and PLA=0.12 mm(3)+/-0.01. Penetrating vessel number: allograft/HBMSC=22.33+/-3.21; allograft=3.67+/-1.153; PLA/HBMSC=32.67+/-8.33; and PLA=7.67+/-3.06. Type 1 collagen and von Willebrand factor immunohistochemistry in scaffold/HBMSC constructs indicated the osteogenic cell phenotype, and new blood vessel formation respectively. Contrast-enhanced 3D reconstructions facilitated the visualization and quantification of neovascularisation. This novel technique has been used to demonstrate neovascularisation in impacted tissue engineered constructs providing a facile approach with wide experimental application

Effect of faradic products on direct current-stimulated calvarial organ culture calcium levels

Calcium release from mouse calvarial organ cultures was used to analyse the well-described biological effects of constant direct current (20 ?A) in combination with Faradic products generated at a titanium wire cathode. Constant 20-?A direct current stimulation alone, delivered by agar salt bridges, consistently lowered the media calcium levels. Direct exposure of calvariae to a titanium cathode and its faradic products resulted in further lowering of media calcium levels and also a significant increase in the media pH. Hydrogen peroxide is a faradic product of the titanium cathode, micromolar amounts being generated by our system over 24 hr. H2O2 is pro-resorptive whereas elevated pH stimulates osteoblast activity. We propose that where bone tissue is in direct contact with metal wire cathodes, the faradic products, hydrogen peroxide and hydroxyl ion, are significant factors which, in their own right, further contribute to accelerated remodelling and improved clinical outcome

Strategies for cell manipulation and skeletal tissue engineering using high-throughput polymer blend formulation and microarray techniques

A combination of high-throughput material formulation and microarray techniques were synergistically applied for the efficient analysis of the biological functionality of 135 binary polymer blends. This allowed the identification of cell-compatible biopolymers permissive for human skeletal stem cell growth in both in vitro and in vivo applications. The blended polymeric materials were developed from commercially available, inexpensive and well characterised biodegradable polymers, which on their own lacked both the structural requirements of a scaffold material and, critically, the ability to facilitate cell growth. Blends identified here proved excellent templates for cell attachment, and in addition, a number of blends displayed remarkable bone-like architecture and facilitated bone regeneration by providing 3D biomimetic scaffolds for skeletal cell growth and osteogenic differentiation. This study demonstrates a unique strategy to generate and identify innovative materials with widespread application in cell biology as well as offering a new reparative platform strategy applicable to skeletal tissue

Supercritical carbon dioxide generated vascular endothelial growth factor encapsulated poly(DL-lactic acid) scaffolds induce angiogenesis in vitro

The ability to deliver, over time, biologically active vascular endothelial growth factor-165 (VEGF) through tailored designed scaffolds offers tremendous therapeutic opportunities to tissue-engineered therapies. Porous biodegradable poly(DL-lactic) acid (PLA) scaffolds encapsulating VEGF have been generated using supercritical CO2 (scCO2) and the kinetic release and angiogenic activity of these scaffolds examined in vitro and in an ex vivo chick chorioallantoic membrane (CAM) angiogenesis model. After processing through scCO2, VEGF maintained its angiogenic activity as assessed by increased tubule formation of human umbilical vein endothelial cells (HUVEC) cultured on Matrigel (VEGF = 1937 +/- 205 microm; scCO2-VEGF = 2085 +/- 234 microm; control = 1237 +/- 179 microm). VEGF release kinetics from scCO2-VEGF incorporated PLA monolith scaffolds showed a cumulative release of VEGF (2837 +/- 761 rhog/ml) over a 21 day period in culture. In addition, VEGF encapsulated PLA scaffolds increased the blood vessel network in the CAM compared to controls; control, 24.8 +/- 9.6; VEGF/PLA, 44.1 +/- 12.1 (vessels/field). These studies demonstrate that the controlled release of growth factors encapsulated into three-dimensional PLA scaffolds can actively stimulate the rapid development of therapeutic neovascularisation to regenerate or engineer tissue

Antibacterial properties of xanthine oxidase in human milk

Formula-fed babies contract gastroenteritis more than breast-fed babies, which is of concern to mothers who cannot breastfeed or, as with HIV-infected mothers, are discouraged from breastfeeding. The ability of endogenous breastmilk xanthine oxidase to generate the antimicrobial radical nitric oxide has been measured and its influence on the growth of Escherichia coli and Salmonella enteritides examined. Breastmilk, but not formula feed, generated nitric oxide. Xanthine oxidase activity substantially inhibited the growth of both bacteria. An important natural antibiotic system is missing in formula feeds; the addition of xanthine oxidase may improve formula for use when breastfeeding is not a safe option.<br/

The application of human bone marrow stromal cells and poly(dl-lactic acid) as a biological bone graft extender in impaction bone grafting

Concerns over disease transmission, high costs and limited supply have led to interest in synthetic grafts in the field of impaction bone grafting (IBG). Poly(DL-lactic acid) (PLA) grafts are attractive alternatives due to their biocompatibility, established safety and versatile manufacturing process. This study examined the potential of PLA scaffolds augmented with human bone marrow stromal cells (HBMSCs) in IBG. In vitro and in vivo studies were performed on impacted morsellised PLA seeded with HBMSC and compared to PLA alone. In vitro samples were incubated under osteogenic conditions and in vivo samples were implanted subcutaneously into severely compromised immunodeficient mice, for 4 weeks. Biochemical, histological, mechanical and 3D micro-computed tomography analyses were performed. HBMSC viability, biochemical activity and histological evidence of osteogenic cellular differentiation, post-impaction were observed in vitro and in vivo in PLA/HBMSC samples compared to impacted PLA alone. In vitro PLA/HBMSC samples demonstrated evidence of mechanical enhancement over PLA alone. In vivo studies showed a significant increase in new bone and blood vessel formation in the PLA/HBMSC constructs compared to PLA alone. With alternatives to allograft being sought, these studies have demonstrated PLA/HBMSC living composites, to be a potential prospect as a biological bone graft extender for future use in the field of IBG