Jason Schense

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.

Enzymatic incorporation of bioactive peptides into fibrin matrices enhances neurite extension

Fibrin plays an important role in wound healing and regeneration, and enjoys widespread use in surgery and tissue engineering. The enzymatic activity of Factor XIIIa was employed to covalently incorporate exogenous bioactive peptides within fibrin during coagulation. Fibrin gels were formed with incorporated peptides from laminin and N-cadherin alone and in combination at concentrations up to 8.2 mol peptide per mole of fibrinogen. Neurite extension in vitro was enhanced when gels were augmented with exogenous peptide, with the maximal improvement reaching 75%. When this particular fibrin derivative was evaluated in rats in the repair of the severed dorsal root within polymeric tubes, the number of regenerated axons was enhanced by 85% relative to animals treated with tubes filled with unmodified fibrin. These results demonstrate that it is possible to enhance the biological activity of fibrin by enzymatically incorporating exogenous oligopeptide domains of morphoregulatory proteins.

Incorporation of heparin-binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering

The goal of this work was to improve the potential of fibrin to promote nerve regeneration by enzymatically incorporating exogenous neurite‐promoting heparin‐binding peptides. The effects on neurite extension of four different heparin‐binding peptides, derived from the heparin‐binding domains of antithrombin III, neural cell adhesion molecule and platelet factor 4, were determined. These exogenous peptides were synthesized as bi‐domain peptide chimeras, with the second domain being a substrate for factor XIIIa. This coagulation transglutaminase covalently bound the peptides within the fibrin gel during coagulation. The heparin‐binding peptides enhanced the degree of neurite extension from embryonic chick dorsal root ganglia through 3‐dimensional fibrin gels, and the extent of enhancement was found to correlate positively with the heparin‐binding affinity of the individual domains. The enhancement could be inhibited by competition with soluble heparin, by degradation of cell‐surface proteoglycans, and by inhibition of the covalent immobilization of the peptide. These results demonstrate an important potential role for proteoglycan‐binding components of the extracellular matrix in neurite extension and suggest that fibrin gels modified with covalently bound heparin‐binding peptides could serve as a therapeutic agent to enhance peripheral nerve regeneration through nerve guide tubes. More generally, the results demonstrate that the biological responses to fibrin, the bodys natural wound healing matrix, can be dramatically improved by the addition of exogenous bioactive peptides in a manner such that they become immobilized during coagulation.—Sakiyama, S. E., Schense, J. C., Hubbell, J. A. Incorporation of heparin‐binding peptides into fibrin gels enhances neurite extension: an example of designer matrices in tissue engineering. FASEB J. 13, 2214–2224 (1999)

Three-dimensional Migration of Neurites Is Mediated by Adhesion Site Density and Affinity

Three-dimensional neurite outgrowth rates within fibrin matrices that contained variable amounts of RGD peptides were shown to depend on adhesion site density and affinity. Bi-domain peptides with a factor XIIIa substrate in one domain and a RGD sequence in the other domain were covalently incorporated into fibrin gels during coagulation through the action of the transglutaminase factor XIIIa, and the RGD-dependent effect on neurite outgrowth was quantified, employing chick dorsal root ganglia cultured two- and three-dimensionally within the modified fibrin. Two separate bi-domain peptides were synthesized, one with a lower binding affinity linear RGD domain and another with a higher binding affinity cyclic RGD domain. Both peptides were cross-linked into fibrin gels at concentrations up to 8.2 mol of peptide/mol of fibrinogen, and their effect on neurite outgrowth was measured. Both two- and three-dimensional neurite outgrowth demonstrated a bi-phasic dependence on RGD concentration for both the linear and cyclic peptide, with intermediate adhesion site densities yielding maximal neurite extension and higher densities inhibiting outgrowth. The adhesion site density that yielded maximal outgrowth depended strongly on adhesion site affinity in both two and three dimensions, with lower densities of the higher affinity ligand being required (0.8–1.7 mol/mol for the linear peptideversus 0.2 mol/mol for the cyclic peptide yielding maximum neurite outgrowth rates in three-dimensional cultures).

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 μg 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 μg/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.

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.

Fibrin biomatrix-conjugated platelet-derived growth factor AB accelerates wound healing in severe thermal injury

Controlled delivery of growth factors from biodegradable biomatrices could accelerate and improve impaired wound healing. The study aim was to determine whether platelet‐derived growth factor AB (PDGF.AB) with a transglutaminase (TG) crosslinking substrate site released from a fibrin biomatrix improves wound healing in severe thermal injury. The binding and release kinetics of TG‐PDGF.AB were determined in vitro. Third‐degree contact burns (dorsum of Yorkshire pigs) underwent epifascial necrosectomy 24 h post‐burn. Wound sites were covered with autologous meshed (3:1) split‐thickness skin autografts and either secured with staples or attached with sprayed fibrin sealant (FS; n = 8/group). TG‐PDGF.AB binds to the fibrin biomatrix using the TG activity of factor XIIIa, and is subsequently released through enzymatic cleavage. Three doses of TG‐PDGF.AB in FS (100 ng, 1 µg and 11 µg/ml FS) were tested. TG‐PDGF.AB was bound to the fibrin biomatrix as evidenced by western blot analysis and subsequently released by enzymatic cleavage. A significantly accelerated and improved wound healing was achieved using sprayed FS containing TG‐PDGF.AB compared to staples alone. Low concentrations (100 ng–1 µg TG‐PDGF.AB/ml final FS clot) demonstrated to be sufficient to attain a nearly complete closure of mesh interstices 14 days after grafting. TG‐PDGF.AB incorporated in FS via a specific binding technology was shown to be effective in grafted third‐degree burn wounds. The adhesive properties of the fibrin matrix in conjunction with the prolonged growth factor stimulus enabled by this binding technology could be favourable in many pathological situations associated with wound‐healing disturbances. Copyright

Mechanical stability in a human radius fracture treated with a novel tissue-engineered bone substitute: a non-invasive, longitudinal assessment using high-resolution pQCT in combination with finite element analysis.

The clinical gold standard in orthopaedics for treating fractures with large bone defects is still the use of autologous, cancellous bone autografts. While this material provides a strong healing response, the use of autografts is often associated with additional morbidity. Therefore, there is a demand for off‐the‐shelf biomaterials that perform similar to autografts. Biomechanical assessment of such a biomaterial in vivo has so far been limited. Recently, the development of high‐resolution peripheral quantitative computed tomography (HR‐pQCT) has made it possible to measure bone structure in humans in great detail. Finite element analysis (FEA) has been used to accurately estimate bone mechanical function from three‐dimensional CT images. The aim of this study was therefore to determine the feasibility of these two methods in combination, to quantify bone healing in a clinical case with a fracture at the distal radius which was treated with a new bone graft substitute. Validation was sought through a conceptional ovine model. The bones were scanned using HR‐pQCT and subsequently biomechanically tested. FEA‐derived stiffness was validated relative to the experimental data. The developed processing methods were then adapted and applied to in vivo follow‐up data of the patient. Our analyses indicated an 18% increase of bone stiffness within 2 months. To our knowledge, this was the first time that microstructural finite element analyses have been performed on bone‐implant constructs in a clinical setting. From this clinical case study, we conclude that HR‐pQCT‐based micro‐finite element analyses show high potential to quantify bone healing in patients. Copyright

Fibrin-based matrices for angio genic stimulation

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