Franz E. Weber

Synthetic matrix metalloproteinase-sensitive hydrogels for the conduction of tissue regeneration: Engineering cell-invasion characteristics

Synthetic hydrogels have been molecularly engineered to mimic the invasive characteristics of native provisional extracellular matrices: a combination of integrin-binding sites and substrates for matrix metalloproteinases (MMP) was required to render the networks degradable and invasive by cells via cell-secreted MMPs. Degradation of gels was engineered starting from a characterization of the degradation kinetics (kcat and Km) of synthetic MMP substrates in the soluble form and after crosslinking into a 3D hydrogel network. Primary human fibroblasts were demonstrated to proteolytically invade these networks, a process that depended on MMP substrate activity, adhesion ligand concentration, and network crosslinking density. Gels used to deliver recombinant human bone morphogenetic protein-2 to the site of critical defects in rat cranium were completely infiltrated by cells and remodeled into bony tissue within 4 wk at a dose of 5 μg per defect. Bone regeneration was also shown to depend on the proteolytic sensitivity of the matrices. These hydrogels may be useful in tissue engineering and cell biology as alternatives for naturally occurring extracellular matrix-derived materials such as fibrin or collagen.

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.

Elucidating the Role of Matrix Stiffness in 3D Cell Migration and Remodeling

Reductionist in vitro model systems which mimic specific extracellular matrix functions in a highly controlled manner, termed artificial extracellular matrices (aECM), have increasingly been used to elucidate the role of cell-ECM interactions in regulating cell fate. To better understand the interplay of biophysical and biochemical effectors in controlling three-dimensional cell migration, a poly(ethylene glycol)-based aECM platform was used in this study to explore the influence of matrix cross-linking density, represented here by stiffness, on cell migration in vitro and in vivo. In vitro, the migration behavior of single preosteoblastic cells within hydrogels of varying stiffness and susceptibilities to degradation by matrix metalloproteases was assessed by time-lapse microscopy. Migration behavior was seen to be strongly dependent on matrix stiffness, with two regimes identified: a nonproteolytic migration mode dominating at relatively low matrix stiffness and proteolytic migration at higher stiffness. Subsequent in vivo experiments revealed a similar stiffness dependence of matrix remodeling, albeit less sensitive to the matrix metalloprotease sensitivity. Therefore, our aECM model system is well suited to unveil the role of biophysical and biochemical determinants of physiologically relevant cell migration phenomena.

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.

IN VIVO AND IN VITRO EVALUATION OF FLEXIBLE, COTTONWOOL-LIKE NANOCOMPOSITES AS BONE SUBSTITUTE MATERIAL FOR COMPLEX DEFECTS

The easy clinical handling and applicability of biomaterials has become a focus of materials research due to rapidly increasing time and cost pressures in the public health sector. The present study assesses the in vitro and in vivo performance of a flexible, mouldable, cottonwool-like nanocomposite based on poly(lactide-co-glycolide) and amorphous tricalcium phosphate nanoparticles (PLGA/TCP 60:40). Immersion in simulated body fluid showed exceptional in vitro bioactivity for TCP-containing fibres (mass gain: 18%, 2 days, HAp deposition). Bone regeneration was quantitatively investigated by creating four circular non-critical-size calvarial defects in New Zealand White rabbits. The defects were filled with the easy applicable cottonwool-like PLGA/TCP fibres or PLGA alone. Porous bovine-derived mineral (Bio-Oss) was used as a positive control and cavities left empty served as a negative control. The area fraction of newly formed bone (4 weeks implantation) was significantly increased for TCP-containing fibres compared to pure PLGA (histological and micro-computed tomographic analysis). A spongiosa-like structure of the newly formed bone tissue was observed for PLGA/TCP nanocomposites, whereas Bio-Oss-treated defects afforded a solid cortical bone.

A randomized-controlled clinical trial evaluating clinical and radiological outcomes after 3 and 5 years of dental implants placed in bone regenerated by means of GBR techniques with or without the addition of BMP-2

OBJECTIVE The aim of this randomized-controlled clinical trial was to evaluate the long-term outcome of implants placed in bone augmented with a xenogenic bone substitute material and a collagen membrane with or without the addition of recombinant human bone morphogenetic protein-2 (rhBMP-2). MATERIAL AND METHODS Eleven patients received a total of 34 implants placed into sites exhibiting lateral bone defects. In a split mouth design, the defects were randomly treated with the graft material and the collagen membrane either with (test) or without (control) rhBMP-2. The patients were examined 3 and 5 years after insertion of the prosthetic restoration. Students paired t-test was performed to detect differences between the two groups. RESULTS The survival rate at 3 and 5 years was 100% for both groups. The peri-implant soft tissues were stable and healthy without any difference between the two groups. The prosthetic reevaluation demonstrated four loose prosthetic screws during the first 3 years and seven ceramic chippings after 3 and 5 years. The mean distance between the first bone to implant contact to implant abutment junction at 3 years was 1.37 mm (test), 1.22 mm (control), and 1.38 mm (test), and 1.23 mm (control) at 5 years. The difference of <0.2 mm between test and control implants was not statistically significant. The mean change of the marginal bone level between baseline and 5 years ranged from -0.07 mm (mesial, test), -0.11 mm (distal, test), -0.03 mm (mesial, control), to +0.13 mm (distal, control). No statistically significant differences were observed between test and control sites. CONCLUSION Implants placed in bone augmented with and without rhBMP-2 revealed excellent clinical and radiological outcomes after 3 and 5 years.

In vitro response of primary human bone marrow stromal cells to recombinant human bone morphogenic protein-2 in the early and late stages of osteoblast differentiation.

A number of factors must be added to human bone marrow stromal cells (hBMSCs) in vitro to induce osteogenesis, including ascorbic acid (AA), β‐glycerophosphate (GP), and dexamethasone (Dex). Bone morphogenic protein (BMP)‐2 is an osteoinductive factor that can commit stromal cells to differentiate into osteoblasts. However, it is still not clear whether the addition of BMP‐2 alone in vitro can induce hBMSCs to complete osteoblast differentiation, resulting in matrix mineralization. This study compares the effects of BMP‐2 and Dex, alone and combined, on the early and late stages of hBMSC differentiation. We found that BMP‐2 causes a significant induction of alkaline phosphatase (ALP) activity in hBMSCs, with a transcriptional upregulation of known BMP‐2‐responsive genes, and the stable expression of cbfa1 in the nucleus and the regions surrounding the nucleus in the early phase of osteoblast differentiation. However, continuous treatment with BMP‐2 alone at doses ranging from 100 to 300 ng/mL results in a less efficient enhancement of in vitro matrix mineralization, despite a significant induction of ALP activity at a concentration of 100 ng/mL. Our results reflect how the effects of BMP‐2 on hBMSCs can vary depending on the stage of osteoblast differentiation, and highlight the need to understand the role of BMP‐2 in primary hBMSCs derived from diverse sources in order to increase the efficiency of using BMP‐2 in osteoinductive therapies.

Engineering 3D cell instructive microenvironments by rational assembly of artificial extracellular matrices and cell patterning.

Engineered artificial microenvironments hold enormous potential as models to study developmental, physiological, pathological, and regenerative processes under highly defined conditions. Such platforms aim at bridging the gap between traditional in vitro 2D culture systems and animal models. By dissecting the biological complexity into an amenable number of parameters, systemic manipulation and study in controllable environments closely resembling the in vivo situation is possible. Novel strategies that address the evaluation of either ECM components, growth factors or cell-cell interactions on cellular behaviour are being developed. However, reliable methods that simultaneously recapitulate the natural instructive microenvironments in terms of cell and matrix composition, biological cues, heterogeneity and geometry are not yet available. Such spatially-defined microenvironments may be necessary to initiate and guide the formation of artificial tissues by morphogenetic processes. In this work, we introduce a flexible strategy that relies on the combination of artificial extracellular matrices with patterning techniques as well as a layer-by-layer approach to mimic rationally-designed instructive milieus. By a rational arrangement of cells and defined biochemical and biophysical extracellular cues, we report control of cell migration and generation of an artificial vascularized bone tissue-like construct.

Biomimetic PEG hydrogels crosslinked with minimal plasmin-sensitive tri-amino acid peptides

Semi-synthetic, proteolytically degradable polymer hydrogels have proven effective as scaffolds to augment bone and skin regeneration in animals. However, high costs due to expensive peptide building blocks pose a significant hurdle towards broad clinical usage of these materials. Here we demonstrate that tri-amino acid peptides bearing lysine (or arginine), flanked by two cysteine residues for crosslinking, are adequate as minimal plasmin-sensitive peptides in poly(ethylene glycol)-based hydrogels formed via Michael-type addition. Substitution of lysine (or arginine) with serine rendered the matrices insensitive to the action of plasmin. This was demonstrated in vitro by performing gel degradation experiments in the presence of plasmin (0.1 U/mL), and in the in vivo situation of regeneration of critical-sized bone defects. When placed as implants into rat calvaria, gels formed from the minimal plasmin substrates showed clear signs of cell infiltration and gel remodeling that coincided with extensive bone formation.

Inhibition of Osteoclast Differentiation and Bone Resorption by N-Methylpyrrolidone

Regulation of RANKL (receptor activator of nuclear factor κB ligand)-induced osteoclast differentiation is of current interest in the development of antiresorptive agents. Osteoclasts are multinucleated cells that play a crucial role in bone resorption. In this study, we investigated the effects of N-methylpyrrolidone (NMP) on the regulation of RANKL-induced osteoclastogenesis. NMP inhibited RANKL-induced tartrate-resistant acid phosphatase activity and the formation of tartrate-resistant acid phosphatase-positive multinucleated cells. The RANKL-induced expression of NFATc1 (nuclear factor of activated T cells, cytoplasmic 1) and c-Fos, which are key transcription factors for osteoclastogenesis, was also reduced by treatment with NMP. Furthermore, NMP induced disruption of the actin rings and decreased the mRNAs of cathepsin K and MMP-9 (matrix metalloproteinase-9), both involved in bone resorption. Taken together, these results suggest that NMP inhibits osteoclast differentiation and attenuates bone resorption. Therefore, NMP could prove useful for the treatment of osteoporosis or other bone diseases associated with excessive bone resorption.