Bone healing induced by local delivery of an engineered parathyroid hormone prodrug.

Regenerative medicine requires innovative therapeutic designs to accommodate high morphogen concentrations in local depots, provide their sustained presence, and enhance cellular invasion and directed differentiation. Here we present an example for inducing local bone regeneration with a matrix-bound engineered active fragment of human parathyroid hormone (PTH(1-34)), linked to a transglutaminase substrate for binding to fibrin as a delivery and cell-invasion matrix with an intervening plasmin-sensitive link (TGplPTH(1-34)). The precursor form displays very little activity and signaling to osteoblasts, whereas the plasmin cleavage product, as it would be induced under the enzymatic influence of cells remodeling the matrix, was highly active. In vivo animal bone-defect experiments showed dose-dependent bone formation using the PTH-fibrin matrix, with evidence of both osteoconductive and osteoinductive bone-healing mechanisms. Results showed that this PTH-derivatized matrix may have potential utility in humans as a replacement for bone grafts or to repair bone defects.

A novel, tissue occlusive poly(ethylene glycol) hydrogel material.

The use of guided bone regeneration (GBR) techniques requires new materials meeting the needs of clinical application. Design criteria for GBR devices are biocompatibility, tissue occlusion, space provision, and clinical manageability. This study evaluates a novel biodegradable poly (ethylene glycol) (PEG) based material as tissue occlusive membrane. A subcutaneous implant model in rats was developed to test the barrier function of the PEG hydrogels over time. Fourteen rats received three membrane implants and two positive controls each. Explants were collected over a period of 7 months. Histological analysis revealed that for at least 4 months cellular infiltration in the membrane explants was lower than 1% of that of the positive controls. Therefore, the PEG based hydrogel can be regarded as tissue occlusive during this period of time. A barrier function seems to be maintained for up to 6 months. In vitro degradation studies performed with the same PEG constructs confirm the in vivo result. In conclusion, our results indicate that this novel PEG-based material has potential for use as a GBR barrier membrane.

Enhanced endothelial cell retention on shear-stressed synthetic vascular grafts precoated with RGD-cross-linked fibrin.

Clinical in vitro endothelialization has been shown to increase the patency of synthetic vascular grafts. The shear stress resistance of the cultured autologous endothelium represents a crucial cornerstone of the concept. We investigated whether an enrichment of the precoating matrix with adhesion sites can augment endothelial cell attachment. Adult human saphenous vein endothelial cells (AHSVECs) were seeded confluently ([58 +/- 11] x 10(3) AHSVECs/cm2) onto 10-cm-long ePTFE (expanded polytetrafluorethylene) vascular grafts (n = 24) precoated with commercial clinically approved fibrin gel (Tisseal) containing various concentrations of cross-linked RGD peptide (0.0, 4.0, 8.0, or 16.0 mg of RGD per milliliter of Tisseal fibrinogen component). Endothelialized grafts were postcultivated for 9 days before they were exposed to a pulsatile circulation model mimicking peak physiological shear stress conditions of the femoral artery (12 dyn/cm2; min/max, -60/+28 dyn/cm2). Cell loss after 24 h was quantitatively determined by image analysis of vital stains. Initial 24-h cell loss was 27.2 +/- 1.7% in grafts precoated with the non-RGD-enriched fibrin matrix. In contrast, cell loss was significantly less on fibrin containing 4.0 mg of RGD peptide per milliliter of Tisseal fibrinogen component (13.3 +/- 7.9%; p < 0.05). Cell loss on fibrin containing 8 and 16 mg of RGD per milliliter of Tisseal fibrinogen component was 41.0 +/- 27.4 and 43.0 +/- 23.2% (p > 0.05), respectively. We conclude that low concentrations of RGD peptide cross-linked into commercial fibrin matrices used for clinical in vitro lining of vascular grafts led to significantly increased endothelial cell retention. The failure of higher RGD concentrations to enhance endothelial cell attachment may be explained by competitive binding of endothelial cells to non-cross-linked RGD.

Bone repair with a form of BMP-2 engineered for incorporation into fibrin cell ingrowth matrices.

Most growth factors naturally involved in development and regeneration demonstrate strong binding to the extracellular matrix and are retained there until being locally mobilized by cells. In spite of this feedback between cell activity and growth factor mobilization in the extracellular matrix, this approach has not been extensively explored in therapeutic situations. We present an engineered bone morphogenetic protein-2 (BMP-2) fusion protein that mimics such function in a surgically relevant matrix, fibrin, incorporated into the matrix until it is locally liberated by cell surface-associated proteases. A tripartite fusion protein, denoted TG-pl-BMP-2, was designed and produced recombinantly. An N-terminal transglutaminase substrate (TG) domain provides covalent attachment to fibrin during coagulation under the influence of the blood transglutaminase factor XIIIa. A central plasmin substrate (pl) domain provides a cleavage site for local release of the attached growth factor from the fibrin matrix under the influence of cell-activated plasmin. A C-terminal human BMP-2 domain provides osteogenic activity. TG-pl-BMP-2 in fibrin was evaluated in vivo in critical-size craniotomy defects in rats, where it induced 76% more defect healing with bone at 3 weeks with a dose of 1 mug/defect than wildtype BMP-2 in fibrin. After a dosing study in rabbits, the engineered growth factor in fibrin was evaluated in a prospective clinical study for pancarpal fusion in dogs, where it induced statistically faster and more extensive bone bridging than equivalent treatment with cancellous bone autograft. The strong healing response shown by fibrin including a bound BMP-2 variant suggests that with the combination of bound growth factor and ingrowth matrix, it may be possible to improve upon the natural growth factor and even upon tissue autograft.

Bone healing in the rat and dog with nonglycosylated BMP-2 demonstrating low solubility in fibrin matrices.

A novel form of recombinant human bone morphogenetic protein-2 (BMP-2) was explored for effective incorporation and long-term retention into fibrin ingrowth matrices. The solubility of native BMP-2 is greatly dependent on its glycosylation. To enhance retention of BMP-2 in fibrin matrices, a nonglycosylated form (nglBMP-2), which is less soluble than the native glycosylated protein, was produced recombinantly and evaluated in critical-size defects in the rat calvarium (group n=6). When 1 or 20 microg nglBMP-2 was incorporated by precipitation within the matrix, 74 +/- 4% and 98 +/- 2% healing was observed in the rat calvarium, respectively, as judged radiographically by closure of the defect at 3 weeks. More soluble forms of BMP-2, used as controls, induced less healing, demonstrating a positive correlation between low solubility, retention in vitro, and healing in vivo. Subsequently, the utility of nglBMP-2 was explored in a prospective veterinary clinical trial for inter-carpal fusion in dogs, replacing the standard-of-care, namely autologous cancellous autograft, with nglBMP-2 in fibrin. In a study of 10 sequential canine patients, fibrin with 600 microg/ml nglBMP-2 performed better than autograft in the first weeks of bone healing and comparably thereafter. Furthermore, a greater fraction of animals treated with nglBMP-2 in fibrin demonstrated bone bridging across each of the treated joints at both 12 and 17 weeks than in animals treated with autograft. These results suggest that evaluation in a human clinical setting of nonglycosylated BMP-2 in fibrin matrices might be fruitful.

Treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 delivered from a fibrin matrix.

OBJECTIVE: To report the results of the treatment of nonunions with nonglycosylated recombinant human bone morphogenetic protein-2 (nglBMP-2) delivered from a designed fibrin matrix. STUDY DESIGN: Experimental trial in rodents and prospective clinical study in dogs and cats with nonunion fractures. ANIMALS: Twenty adult female, albino, Sprague-Dawley rats; 8 client-owned cats and dogs. METHODS: After development of a fibrin matrix and evaluation of nglBMP-2 in a rodent femoral defect model, 8 consecutive long bone nonunion fractures (no progression in healing in > or = 3 months), were treated using 300 microg nglBMP-2 in a liquid fibrin precursor, injected into the defect gap after fracture revision and stabilization, or through a stab incision into the fracture site. The fibrin matrix was designed to clot in the wound after 60 seconds and to release the nglBMP-2 continuously over several days. RESULTS: Using only fibrin gel, 7% of the rat femoral defect was filled with new formed bone compared with 79% defect filling using 2 microg nglBMP-2 (P=.006). Five and 10 microg nglBMP in fibrin resulted in union of all femoral defects with complete filling of the gap with new bone. Bony bridging and clinical healing was achieved in 7 patients within 24 weeks of administration of nglBMP-2. CONCLUSIONS: Application of nglBMP-2 in a functional matrix can induce bone healing. Controlled release of nglBMP-2 from a fibrin matrix mimics the natural fracture hematoma. CLINICAL RELEVANCE: nglBMP-2/fibrin can successfully replace a cancellous bone autograft in fracture treatment with an associated reduction in graft donor site morbidity and surgical time.

Repair of bone defects using synthetic mimetics of collagenous extracellular matrices.

We have engineered synthetic poly(ethylene glycol) (PEG)-based hydrogels as cell-ingrowth matrices for in situ bone regeneration. These networks contain a combination of pendant oligopeptide ligands for cell adhesion (RGDSP) and substrates for matrix metalloproteinase (MMP) as linkers between PEG chains. Primary human fibroblasts were shown to migrate within these matrices by integrin- and MMP-dependent mechanisms. Gels used to deliver recombinant human bone morphogenetic protein-2 (rhBMP-2) to the site of critical- sized defects in rat crania were completely infiltrated by cells and were remodeled into bony tissue within five weeks. Bone regeneration was dependent on the proteolytic sensitivity of the matrices and their architecture. The cell-mediated proteolytic invasiveness of the gels and entrapment of rhBMP-2 resulted in efficient and highly localized bone regeneration.