Dror Ben-David

Engineering the Growth Factor Microenvironment with Fibronectin Domains to Promote Wound and Bone Tissue Healing

A multifunctional fibronectin fragment enhances the regenerative effects of growth factors in vivo in animal models of chronic wounds and critical-size bone defects. Sweet Synergy Engineers have long been interested in creating the perfect environment for repairing injured tissues, which range from broken blood vessels to shattered nerves. Such efforts have included both simple materials, like collagen, and complex ones comprising a polymeric labyrinth of biomolecules and cells. As described in this issue, Martino et al. have hit the sweet spot for engineering the cellular microenvironment: a combination of natural polymer and recombinant protein that recruits growth factors to wounds and convinces cells to repair the damage. Martino and colleagues sought to generate a matrix that would sequester growth factors. The authors started with a fibrin matrix, which is used clinically as a tissue substitute to promote healing. Next, they pieced together two fibronectin (FN) fragments—the 9th to 10th and the 12th to 14th type III repeats—to control integrin and growth factor binding, respectively. Finally, the resulting recombinant FN fragment, FN III9-10/12-14, was covalently immobilized on the fibrin scaffold. The FN III9-10/12-14 matrix was able to bind vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and bone morphogenetic protein (BMP)—three factors that are intricately involved in skin and bone repair. FN III9-10/12-14 in combination with VEGF and PDGF enhanced proliferation of endothelial cells, smooth muscle cells, and mesenchymal stem cells in vitro. The engineered FN fragment, when co-delivered with all three growth factors, also stimulated cell migration to a greater extent than control FN proteins, suggesting improved signaling synergy between growth factors and the recombinant FN. To see whether the material healed tissues in vivo, Martino and colleagues injected their designer scaffold into the wounds of diabetic mice and into the calvarial defects of skeletally mature rats. Enhanced reepithelialization, granulation tissue formation, and angiogenesis were noted for the wounds. For the bone defects, the authors reported increased bone tissue deposition and recruitment of bone progenitor cells. These preclinical demonstrations in rodent models show promise for the use of the FN III9-10/12-14–modified matrices in humans to heal chronic wounds and repair bones. Although testing in larger animal models might be necessary before translation, it is clear that these engineered microenvironments improve the synergy between endogenous growth factors and cells to restore tissue form and function to normal. Although growth factors naturally exert their morphogenetic influences within the context of the extracellular matrix microenvironment, the interactions among growth factors, their receptors, and other extracellular matrix components are typically ignored in clinical delivery of growth factors. We present an approach for engineering the cellular microenvironment to greatly accentuate the effects of vascular endothelial growth factor–A (VEGF-A) and platelet-derived growth factor–BB (PDGF-BB) for skin repair, and of bone morphogenetic protein–2 (BMP-2) and PDGF-BB for bone repair. A multifunctional recombinant fragment of fibronectin (FN) was engineered to comprise (i) a factor XIIIa substrate fibrin-binding sequence, (ii) the 9th to 10th type III FN repeat (FN III9-10) containing the major integrin-binding domain, and (iii) the 12th to 14th type III FN repeat (FN III12-14), which binds growth factors promiscuously, including VEGF-A165, PDGF-BB, and BMP-2. We show potent synergistic signaling and morphogenesis between α5β1 integrin and the growth factor receptors, but only when FN III9-10 and FN III12-14 are proximally presented in the same polypeptide chain (FN III9-10/12-14). The multifunctional FN III9-10/12-14 greatly enhanced the regenerative effects of the growth factors in vivo in a diabetic mouse model of chronic wounds (primarily through an angiogenic mechanism) and in a rat model of critical-size bone defects (through a mesenchymal stem cell recruitment mechanism) at doses where the growth factors delivered within fibrin only had no significant effects.

The innate osteogenic potential of the maxillary sinus (Schneiderian) membrane: an ectopic tissue transplant model simulating sinus lifting.

Maxillary sinus membrane lifting is a common procedure aimed at increasing the volume of the maxillary sinus osseous floor prior to inserting dental implants. Clinical observations of bone formation in sinus lifting procedures without grafting bone substitutes were observed, but the biological nature of bone regeneration in sinus lifting procedures is unclear. This study tested whether this osteogenic activity relies on inherent osteogenic capacity residing in the sinus membrane by simulating the in vivo clinical condition of sinus lifting in an animal model. Maxillary sinus membrane cells were cultured in alpha-MEM medium containing osteogenic supplements (ascorbic acid, dexamethasone). Cultured cells revealed alkaline phosphatase activity and mRNA expression of osteogenic markers (alkaline phosphatase, bone sialoprotein, osteocalcin and osteonectin) verifying the osteogenic potential of the cells. Fresh tissue samples demonstrated positive alkaline phosphatase enzyme activity situated along the membrane-bone interface periosteum-like layer. To simulate the in vivo clinical conditions, the membranes were folded to form a pocket-like structure and were transplanted subcutaneously in immunodeficient mice for 8 weeks. New bone formation was observed in the transplants indicating the innate osteogenic potential within the maxillary Schneiderian sinus membrane and its possible contribution to bone regeneration in sinus lifting procedures.

Controlled release of BMP-2 from a sintered polymer scaffold enhances bone repair in a mouse calvarial defect model.

Sustained and controlled delivery of growth factors, such as bone morphogenetic protein 2 (BMP‐2), from polymer scaffolds has excellent potential for enhancing bone regeneration. The present study investigated the use of novel sintered polymer scaffolds prepared using temperature‐sensitive PLGA/PEG particles. Growth factors can be incorporated into these scaffolds by mixing the reconstituted growth factor with the particles prior to sintering. The ability of the PLGA/PEG scaffolds to deliver BMP‐2 in a controlled and sustained manner was assessed and the osteogenic potential of these scaffolds was determined in a mouse calvarial defect model. BMP‐2 was released from the scaffolds in vitro over 3 weeks. On average, ca. 70% of the BMP‐2 loaded into the scaffolds was released by the end of this time period. The released BMP‐2 was shown to be active and to induce osteogenesis when used in a cell culture assay. A substantial increase in new bone volume of 55% was observed in a mouse calvarial defect model for BMP‐2‐loaded PLGA/PEG scaffolds compared to empty defect controls. An increase in new bone volume of 31% was observed for PLGA/PEG scaffolds without BMP‐2, compared to empty defect controls. These results demonstrate the potential of novel PLGA/PEG scaffolds for sustained BMP‐2 delivery for bone‐regeneration applications. Copyright

Low dose BMP-2 treatment for bone repair using a PEGylated fibrinogen hydrogel matrix

Bone repair strategies utilizing resorbable biomaterial implants aim to stimulate endogenous cells in order to gradually replace the implant with functional repair tissue. These biomaterials should therefore be biodegradable, osteoconductive, osteoinductive, and maintain their integrity until the newly formed host tissue can contribute proper function. In recent years there has been impressive clinical outcomes for this strategy when using osteoconductive hydrogel biomaterials in combination with osteoinductive growth factors such as human recombinant bone morphogenic protein (hrBMP-2). However, the success of hrBMP-2 treatments is not without risks if the factor is delivered too rapidly and at very high doses because of a suboptimal biomaterial. Therefore, the aim of this study was to evaluate the use of a PEGylated fibrinogen (PF) provisional matrix as a delivery system for low-dose hrBMP-2 treatment in a critical size maxillofacial bone defect model. PF is a semi-synthetic hydrogel material that can regulate the release of physiological doses of hrBMP-2 based on its controllable physical properties and biodegradation. hrBMP-2 release from the PF material and hrBMP-2 bioactivity were validated using in vitro assays and a subcutaneous implantation model in rats. Critical size calvarial defects in mice were treated orthotopically with PF containing 8 μg/ml hrBMP-2 to demonstrate the capacity of these bioactive implants to induce enhanced bone formation in as little as 6 weeks. Control defects treated with PF alone or left empty resulted in far less bone formation when compared to the PF/hrBMP-2 treated defects. These results demonstrate the feasibility of using a semi-synthetic biomaterial containing small doses of osteoinductive hrBMP-2 as an effective treatment for maxillofacial bone defects.

Cell-scaffold transplant of hydrogel seeded with rat bone marrow progenitors for bone regeneration

Bone is the second most frequently transplanted tissue in humans and efforts are focused on developing cell-scaffold constructs which can be employed for autologous implantation in place of allogenic transplants. The objective of the present study was to examine the efficacy of a gelatin-based hydrogel scaffold to support osteogenic differentiation of rat bone marrow-derived mesenchymal stem cells (MSCs) and its application in a cranial defect model. MSCs which were cultured on hydrogel under osteogenic conditions demonstrated typical osteogenic differentiation which included cluster formation with positive Alizarin Red S staining, sedimentation of calcium phosphate as defined by SEM and EDS spectroscopy and expression of mRNA osteogenic markers. Empty scaffolds or those containing either differentiated cells or naïve cells were implanted into cranial defects of athymic nude mice and the healing process was followed by μCT. Substantial bone formation (65%) was observed with osteogenic cell-scaffold constructs when compared to the naïve cell construct (25%) and the cell free scaffold (10%). Results demonstrated the potential of hydrogel scaffolds to serve as a supportive carrier for bone marrow-derived MSCs.

A tissue‐like construct of human bone marrow MSCs composite scaffold support in vivo ectopic bone formation

Biocompatible and osteoconductive cell–scaffold constructs comprise the first and most important step towards successful in vivo bone repair. This study reports on a new cell–scaffold construct composed of gelatin‐based hydrogel and ceramic (CaCO3/β‐TCP) particles loaded with human MSCs producing a tissue‐like construct applied as a transplant for in vivo bone formation. Bone marrow‐derived human MSCs were cultured in osteogenic induction medium. 5 × 105 (P2) cells were loaded on a mixture of hydrogel microspheres and ceramic particles, cultured in a rotating dynamic culture for up to 3 weeks. Both hydrogel microspheres and ceramic particles coalesced together to form a tissue‐like construct, shown by histology to contain elongated spindle‐like cells forming the new tissue between the individual particles. Cell proliferation and cell viability were confirmed by Alamar blue assay and by staining with CFDA, respectively. FACS analysis conducted before loading the cells, and after formation of the construct, revealed that the profile of cell surface markers remained unchanged throughout the dynamic culture. The osteogenic potential of the cells composing the tissue‐like construct was further validated by subcutaneous transplants in athymic nude mice. After 8 weeks a substantial amount of new bone formation was observed in the cell‐construct transplants, whereas no bone formation was observed in transplants containing no cells. This new cell construct provides a system for in vivo bone transplants. It can be tailored for a specific size and shape as needed for various transplant sites and for all aspects of regenerative medicine and biomaterial science. Copyright

Lentiviral-Mediated Integrin α5 Expression in Human Adult Mesenchymal Stromal Cells Promotes Bone Repair in Mouse Cranial and Long-Bone Defects

Abstract Adult human mesenchymal stromal cells (hMSCs) are an important source for tissue repair in regenerative medicine. Notably, targeted gene therapy in hMSCs to promote osteogenic differentiation may help in the development of novel therapeutic approaches for bone repair. We recently showed that α5 integrin (ITGA5) promotes osteoblast differentiation in bone marrow-derived hMSCs. Here, we determined whether lentiviral (LV)-mediated expression of ITGA5 in hMSCs derived from the bone-marrow stroma of healthy individuals may promote bone repair in vivo in two relevant critical-size bone defects in the mouse. In a first series of experiments, control or LV-ITGA5-transduced hMSCs were seeded on collagen-based gelatin sponge and transplanted in a cranial critical-size defect (5 mm) in Nude-Foxn1nu mice. Microcomputed tomography and quantitative histological analyses after 8 weeks showed no or little de novo bone formation in defects implanted with collagen sponge alone or with hMSCs, respectively. In contrast, implantation of collagen sponge with LV-ITGA5-transduced hMSCs showed greater bone formation compared with control hMSCs. We also tested the bone-repair potential of LV-mediated ITGA5 expression in hMSCs in a critical-size long-bone defect (2 mm) in femur in Nude-Foxn1nu mice. Bone remnants were stabilized with external fixation, and control or LV-ITGA5-transduced hMSCs mixed with coral/hydroxyapatite particles were transplanted into the critical-size long-bone defect. Histological analysis after 8 weeks showed that LV-ITGA5-transduced hMSCs implanted with particles induced 85% bone regeneration and repair. These results demonstrate that repair of critical-size mouse cranial and long-bone defects can be induced using LV-mediated ITGA5 gene expression in hMSCs, which provides a novel gene therapy for bone regeneration.

Evaluation of the osteoconductive potential of bone substitutes embedded with schneiderian membrane‐ or maxillary bone marrow‐derived osteoprogenitor cells

AIM Sinus augmentation procedures commonly employ osteoconductive scaffolding materials to stimulate and support bone formation. The aim of this study was to develop a simple screening methodology for the evaluation of the osteoconductive potential of various bone graft materials prior to clinical use. MATERIALS AND METHODS Materials tested were Bio-Oss, Bi-Ostetic, OraGraft, and ProOsteon. These Simple and composite bone substitutes were embedded with osteoprogenitor cells derived from either the human maxillary sinus schneiderian membrane (hMSSM) or from maxillary tuberosity bone marrow and then monitored both in vitro and in vivo. RESULTS Cell adherence and proliferation was most pronounced in OraGraft, followed by ProOsteon. In vivo bone formation, within the bone graft, was also observed, with most marked results in OraGraft and ProOsteon grafts. CONCLUSIONS The proposed osteoconductivity testing method proved simple, informative, and reliable for the purpose of screening candidate biomaterials for sinus lifting or sinus augmentation.

Engineering vascularized flaps using adipose-derived microvascular endothelial cells and mesenchymal stem cells

Human adipose‐derived microvascular endothelial cells (HAMEC) and mesenchymal stem cells (MSC) have been shown to bear angiogenic and vasculogenic capabilities. We hypothesize that co‐culturing HAMEC:MSC on a porous biodegradable scaffold in vitro, later implanted as a graft around femoral blood vessels in a rat, will result in its vascularization by host vessels, creating a functional vascular flap that can effectively treat a range of large full‐thickness soft tissue defects. HAMEC were co‐cultured with MSC on polymeric three‐dimensional porous constructs. Grafts were then implanted around the femoral vessels of a rat. To ensure vessel sprouting from the main femoral vessels, grafts were pre‐isolated from the surrounding tissue. Graft vascularization was monitored to confirm full vascularization before flap transfer. Flaps were then transferred to treat both abdominal wall and exposed bone and tendon of an ankle defects. Flaps were analysed to determine vascular properties in terms of maturity, functionality and survival of implanted cells. Findings show that pre‐isolated grafts bearing the HAMEC:MSC combination promoted formation of highly vascularized flaps, which were better integrated in both defect models. The results of this study show the essentiality of a specific adipose‐derived cell combination in successful graft vascularization and integration, two processes crucial for flap survival. Copyright

The involvement of oxidants and NF-κB in cytokine-induced MMP-9 synthesis by bone marrow-derived osteoprogenitor cells.

Objective and designThe activity of immune cells affects the balance between bone mineralization and resorption carried out by the opposing actions of osteoblasts and osteoclasts, respectively. This study was aimed at determining the possible interaction between inflammatory conditions and collagen type I degrading MMP (mainly MMP-2 and MMP-9) synthesis and secretion in rat osteoprogenitors.Materials and methodsThe study was performed using primary rat bone marrow-derived osteoprogenitors during their advanced osteogenesis. Biochemical, immunohistochemical, and molecular biology techniques were used to investigate the influence of pro-inflammatory cytokines on MMP-2 and MMP-9 synthesis and secretion in osteoprogenitors.ResultsResults indicated that both synthesis and secretion of MMPs (MMP-1, -2, -8, -9, and -13) were significantly induced after pro-inflammatory cytokine treatments, except MMP-2, whose levels remained unchanged. NF-κB (nuclear factor kappa-light chain enhancer of activated B cells) inhibition assays showed that induced MMP-9 secretion by inflammatory cytokines was mediated by activation of NF-κB via the classical pathway and that oxidants play a significant role in this signal transduction pathway. In contrast, no such effect was observed for synthesis of MMP-2.ConclusionsThese results indicate the possibility that inflammatory processes may trigger osteoblasts to absorb bone by secreting elevated levels of MMPs capable of degrading collagen type I, especially MMP-9 which is upregulated due to increased NF-κB transcription activity.