Staffan Dånmark

A strategy for the covalent functionalization of resorbable polymers with heparin and osteoinductive growth factor

The chemical strategy presented herein is the nondestructive preparation of resorbable polymer scaffolds with heparin covalently bonded to the surface and an osteoinductive growth factor, recombinant human bone morphogenetic protein-2, immobilized in the heparin layer. The coupling scheme involves functionalization of surfaces by grafting in the vapor phase with poly( l-lactide) and poly(-caprolactone) films chosen as representative substrates. The biocompatibility of functionalized surfaces was verified by a much improved attachment and proliferation of mesenchymal stem cells (MSC).

Osteogenic Differentiation by Rat Bone Marrow Stromal Cells on Customized Biodegradable Polymer Scaffolds

In this report, poly(L-lactide-co-ε-caprolactone), poly(LLA-co-CL) and poly(L-lactide-co-1,5-dioxepan-2-one), poly(LLA-co-DXO) were evaluated and compared for potential use in bone tissue engineering constructs together with bone marrow stromal cells (BMSC). The copolymers were tailored to reduce the level of harmful tin residuals in the scaffolding. BMSC isolated from Sprague—Dawley rats were seeded onto the scaffolds and cultured in vitro for up to 21 days. Cell spreading and proliferation was analyzed after 72 h by scanning electron microscopy and thiazolyl blue tetrazolium bromide (MTT) conversion assay. Osteogenic differentiation of BMSC was evaluated by real-time PCR after 14 and 21 days of culture. Hydrophilicity was significantly different between poly(LLA-co-CL) and poly(LLA-co-DXO) with the latter being more hydrophilic. After 72 h, both scaffolds supported increased cell proliferation and the mRNA expression of osteocalcin and osteopontin was significantly increased after 21 days. Further investigation of these constructs, with lower levels of tin residuals, are being pursued.

In vitro and in vivo degradation profile of aliphatic polyesters subjected to electron beam sterilization

Degradation characteristics in response to electron beam sterilization of designed and biodegradable aliphatic polyester scaffolds are relevant for clinically successful synthetic graft tissue regeneration. Scaffold degradation in vitro and in vivo were documented and correlated to the macroscopic structure and chemical design of the original polymer. The materials tested were of inherently diverse hydrophobicity and crystallinity: poly(L-lactide) (poly(LLA)) and random copolymers from L-lactide and ε-caprolactone or 1,5-dioxepan-2-one, fabricated into porous and non-porous scaffolds. After sterilization, the samples underwent hydrolysis in vitro for up to a year. In vivo, scaffolds were surgically implanted into rat calvarial defects and retrieved for analysis after 28 and 91days. In vitro, poly(L-lactide-co-1,5-dioxepan-2-one) (poly(LLA-co-DXO)) samples degraded most rapidly during hydrolysis, due to the pronounced chain-shortening reaction caused by the sterilization. This was indicated by the rapid decrease in both mass and molecular weight of poly(LLA-co-DXO). Poly(L-lactide-co-ε-caprolactone) (poly(LLA-co-CL)) samples were also strongly affected by sterilization, but mass loss was more gradual; molecular weight decreased rapidly during hydrolysis. Least affected by sterilization were the poly(LLA) samples, which subsequently showed low mass loss rate and molecular weight decrease during hydrolysis. Mechanical stability varied greatly: poly(LLA-co-CL) withstood mechanical testing for up to 182 days, while poly(LLA) and poly(LLA-co-DXO) samples quickly became too brittle. Poly(LLA-co-DXO) samples unexpectedly degraded more rapidly in vitro than in vivo. After sterilization by electron beam irradiation, the three biodegradable polymers present widely diverse degradation profiles, both in vitro and in vivo. Each exhibits the potential to be tailored to meet diverse clinical tissue engineering requirements.

Resveratrol-conjugated poly-ε-caprolactone facilitates in vitro mineralization and in vivo bone regeneration

Incorporation of osteoinductive factors in a suitable scaffold is considered a promising strategy for generating osteogenic biomaterials. Resveratrol is a polyphenol found in parts of certain plants, including nuts, berries and grapes. It is known to increase DNA synthesis and alkaline phosphatase (ALP) activity in osteoblasts and to prevent femoral bone loss in ovariectomized (OVX) rats. In the present study resveratrol was coupled through a hydrolysable covalent bond with the carboxylic acid groups in porous poly-ε-caprolactone (PCL) surface grafted with acrylic acid (AA). The osteogenic effect of this new scaffold was evaluated in mesenchymal cell culture and in the rat calvarial defect model. We found that the incorporation of resveratrol caused increased ALP activity of rat bone marrow stromal cells and enhanced mineralization of the cell-scaffold composites in vitro. After 8 weeks the calvarial defects implanted with resveratrol-conjugated PCL displayed a higher X-ray density than the defects implanted with control PCL. Bone-like structures, positively immunostained for bone sialoprotein, were shown to be more extensively formed in the resveratrol-conjugated PCL. These results show that incorporation of resveratrol into the AA-functionalized porous PCL scaffold led to a significant increase in osteogenesis.

Biocompatibility of Polyester Scaffolds with Fibroblasts and Osteoblast-like Cells for Bone Tissue Engineering

The aim of this study was to evaluate the in vitro cytotoxicity and cytocompatibility of the developed aliphatic polyester co-polymer scaffolds: poly(L-lactide-co-ε-caprolactone) and poly(L-lactide-co-1,5-dioxepan-2-one). The scaffolds were produced by solvent casting and particulate leaching, and tested by direct and indirect contact cytotoxicity assays on human osteoblast-like cells and mouse fibroblasts. Cell morphology was documented by light and scanning electron microscopy. Viability was assessed by the MTT, neutral red uptake, lactic dehydrogenase and apoptosis assays. Extraction tests confirmed that the scaffolds did not have a cytotoxic effect on the cells. The cells grew and spread well on the test scaffolds with good cellular attachment and viability. The scaffolds are noncytotoxic and biocompatible with the two cell types and warrant continued investigation as potential constructs for bone tissue engineering.

Effect of endothelial cells on bone regeneration using poly(L-lactide-co-1,5-dioxepan-2-one) scaffolds

Our recent in vitro study demonstrated that endothelial cells (ECs) might influence the differentiation of bone marrow stromal cells (BMSCs). Therefore, the aim of this study was to describe this effect in vivo, using a rat calvarial bone defect model. BMSCs were isolated from femurs of two-donor Lewis rats and expanded in α-minimum essential medium containing 10% fetal bovine serum. One fifth of BMSCs were induced and differentiated into ECs in an Endothelial Cell Growth Medium-2 and then characterized by a flow cytometry. The remaining BMSCs were cultured in freshly prepared osteogenic stimulatory medium, containing dexamethasone, ascorbic acid and β-glycerophosphate. Either BMSCs alone (BMSC-group) or co-cultured ECs/BMSCs (CO-group) were seeded into poly(L-lactide-co-1,5-dioxepan-2-one) [poly(LLA-co-DXO)] scaffolds, cultured in spinner flasks, and then implanted into symmetrical calvarial defects prepared in recipient rats. The animals were sacrificed after 2 months. The formation of new bone was evaluated by radiography and histology and by the expression of osteogenic markers using reverse transcriptase-polymerized chain reaction (RT-PCR). To investigate vessel formation, histological staining was performed with ECs markers. The radiographical and histological results showed more rapid bone formation in the CO- than in the BMSC-group. However, the expression of ECs marker was similar on both groups by histological analysis after 2 months postoperatively. Furthermore, the CO-group exhibited greater expression of osteogenic markers as demonstrated by RT-PCR. The results are consistent with the previous in vitro findings that poly(LLA-co-DXO) scaffold might be suitable candidate for bone tissue engineering. In vivo, bone regeneration was enhanced by a construct of the polymer scaffold loaded with co-cultured cells.

Growth and differentiation of bone marrow stromal cells on biodegradable polymer scaffolds : An in vitro study

A fundamental component of bone tissue engineering is an appropriate scaffold as a carrier for osteogenic cells. The aim of the study was to evaluate the response of human bone marrow stromal cells (BMSC) to scaffolds made of three biodegradable polymers: poly(L-lactide-co-ε-caprolactone) (poly(LLA-co-CL)), poly(L-lactide-co-1,5dioxepan-2-one) (poly(LLA-co-DXO)), and poly(L-lactide) (poly(LLA)). Cellular response was evaluated in terms of attachment, proliferation, and differentiation. SEM disclosed earlier cell attachment and better spreading on poly(LLA-co-CL) and poly(LLA-co-DXO) scaffolds than on poly(LLA) after 1 h. At 24 h and 14 days postseeding, BMSCs had spread well, forming multiple cellular layers on the scaffolds. Cell proliferation was higher on poly(LLA-co-CL) and on poly(LLA-co-DXO) than on poly(LLA) after 1 and 7 days. Cell growth cycles of BMSC were longer on the scaffolds than on coverslips. After 7 and 14 days cultivation on scaffolds, the expression of osteogenic markers such as ALP, Col I, OPN, and Runx2 were stimulated by BMSC, which indicating that poly(LLA-co-DXO), poly(LLA-co-CL), and poly(LLA) could support the osteogenic differentiation of BMSC in vitro. Poly(LLA-co-CL) and poly(LLA-co-DXO) promoted better attachment and growth of BMSC than poly(LLA). BMSC also retained their osteogenic differentiation potential, indicating biological activity of BMSC on the scaffolds. The promising results of this in vitro study indicate that these copolymers warrant further evaluation for potential application in bone tissue engineering.

Self-sorting heterodimeric coiled coil peptides with defined and tuneable self-assembly properties

Coiled coils with defined assembly properties and dissociation constants are highly attractive components in synthetic biology and for fabrication of peptide-based hybrid nanomaterials and nanostructures. Complex assemblies based on multiple different peptides typically require orthogonal peptides obtained by negative design. Negative design does not necessarily exclude formation of undesired species and may eventually compromise the stability of the desired coiled coils. This work describe a set of four promiscuous 28-residue de novo designed peptides that heterodimerize and fold into parallel coiled coils. The peptides are non-orthogonal and can form four different heterodimers albeit with large differences in affinities. The peptides display dissociation constants for dimerization spanning from the micromolar to the picomolar range. The significant differences in affinities for dimerization make the peptides prone to thermodynamic social self-sorting as shown by thermal unfolding and fluorescence experiments, and confirmed by simulations. The peptides self-sort with high fidelity to form the two coiled coils with the highest and lowest affinities for heterodimerization. The possibility to exploit self-sorting of mutually complementary peptides could hence be a viable approach to guide the assembly of higher order architectures and a powerful strategy for fabrication of dynamic and tuneable nanostructured materials.

Integrin-mediated adhesion of human mesenchymal stem cells to extracellular matrix proteins adsorbed to polymer surfaces

In vitro, degradable aliphatic polyesters are widely used as cell carriers for bone tissue engineering, despite their lack of biological cues. Their biological active surface is rather determined by an adsorbed layer of proteins from the surrounding media. Initial cell fate, including adhesion and proliferation, which are key properties for efficient cell carriers, is determined by the adsorbed layer of proteins. Herein we have investigated the ability of human bone marrow derived stem cells (hBMSC) to adhere to extracellular matrix (ECM) proteins, including fibronectin and vitronectin which are present in plasma and serum. hBMSC expressed integrins for collagens, laminins, fibronectin and vitronectin. Accordingly, hBMSC strongly adhered to these purified ECM proteins by using the corresponding integrins. Although purified fibronectin and vitronectin adsorbed to aliphatic polyesters to a lower extent than to cell culture polystyrene, these low levels were sufficient to mediate adhesion of hBMSC. It was found that plasma- and serum-coated polystyrene adsorbed significant levels of both fibronectin and vitronectin, and fibronectin was identified as the major adhesive component of plasma for hBMSC; however, aliphatic polyesters adsorbed minimal levels of fibronectin under similar conditions resulting in impaired cell adhesion. Altogether, the results suggest that the efficiency of aliphatic polyesters cell carriers could be improved by increasing their ability to adsorb fibronectin.

Comparison of short-run cell seeding methods for poly(L-lactide-co-1,5-dioxepan-2-one) scaffold intended for bone tissue engineering

Constructs intended for bone tissue engineering are influenced by the initial cell seeding procedure. The seeding method should be rapid, convenient, improve cell spatial distribution, and have no negative effects on cellular viability and differentiation. This study aimed to compare the effect of short-run seeding methods (centrifuge and vortex) with a static method on the scaffolds prepared from poly(L-lactide-co-1,5-dioxepan-2-one) by solvent-casting particulate-leaching (SCPL) technique. Human osteoblast-like cells (HOB) were seeded by the three methods described above. The seeding efficiency was determined by attached cell numbers. Cellular proliferation was analyzed by WST-1 and dsDNA assay. Cell distribution was examined by scanning electron (SEM) and fluorescence microscopy. Expression of Alkaline Phosphatase (ALP), Collagen type I (Col I), Osteocalcin (OC) and Proliferating Cell Nuclear Antigen (PCNA) were determined by real time RT-PCR. Results indicated that centrifuge and vortex increased seeding efficiency and had no negative effects on cellular viability. The data obtained by the fluorescence microscope confirmed the SEM results that the vortex method improved cell distribution through the scaffolds more than the other two methods (p<0.05). The RT-PCR results showed no significant differences on the expression of mRNA between the three methods of the above markers. The vortex method was found to be a simple and feasible seeding method for the poly(L-lactide-co-1,5-dioxepan-2-one) scaffolds.