Susanne Bierbaum

Modifications of hyaluronan influence the interaction with human bone morphogenetic protein-4 (hBMP-4).

In this study, we have demonstrated that the modification of hyaluronan (hyaluronic acid; Hya) with sulfate groups led to different binding affinities for recombinant human bone morphogenetic protein-4 (rhBMP-4). The high-sulfated sHya2.8 (average degree of sulfation (D.S.) 2.8) exhibited the tightest interaction with rhBMP-4, followed by the low-sulfated sHya1.0, as determined with surface plasmon resonance (SPR), ELISA, and competition ELISA. Unmodified Hya, chondroitin-sulfate (CS), and heparan sulfate (HS) showed significantly less binding affinity. SPR data could be fitted to an A + B = AB Langmuir model and binding constants were evaluated ranging from 13 pM to 5.45 microM. The interaction characteristics of the differentially sulfated Hyas are promising for the incorporation of these modified polysaccharides in bioengineered coatings of biomaterials for medical applications.

Effect of modifications of dual acid‐etched implant surfaces on peri‐implant bone formation. Part I: organic coatings

OBJECTIVE The aim of the present study was to test the hypothesis that peri-implant bone formation can be improved by modifying dual acid-etched (DAE) implant surfaces using organic coatings that enhance cell adhesion and osteogenic differentiation. MATERIAL AND METHODS Ten adult female foxhounds received experimental titanium implants in the mandible 3 months after removal of all premolar teeth. Six types of implants were evaluated in each animal: (i) implants with a machined surface (MS), (ii) implants with a DAE surface topography, (iii) implants with an acid-etched surface coated with RGD peptides, (iv) implants with an acid-etched surface coated with collagen I, (v) implants with an acid-etched surface coated with collagen I and chondroitin sulphate (CS), (vi) implants with an acid-etched surface coated with collagen I and CS and recombinant human bone morphogenetic protein-2. Peri-implant bone regeneration was assessed by histomorphometry after 1 and 3 months in five dogs each by measuring bone implant contact (BIC) and the bone volume density (BVD) of the newly formed peri-implant bone. RESULTS After 1 month, mean BIC was significantly higher in the coated implants group than in the MS group. There was no significant difference when mean BIC in the DAE group was compared with implants with any of the organic coatings, but the difference was significant when compared with the MS implants. Differences in mean BVD value did not reach significance between any of the surfaces. After 3 months, the same held true for the mean BIC of all the groups except for Coll I. Mean volume density of the newly formed bone was higher in all the surface modifications, albeit without statistical significance. CONCLUSIONS It is concluded that with the exception of Coll I, the tested organic surface coatings on DAE surfaces did not improve peri-implant bone formation when compared with the DAE surfaces but enhanced BIC when compared with the MSs.

Biological functionalization of dental implants with collagen and glycosaminoglycans—A comparative study

Biological implant surface coatings are an emerging technology to increase bone formation. Such an approach is of special interest in anatomical regions like the maxilla. In the present study, we hypothesized that the coating of titanium implants with components of the organic extracellular matrix increases bone formation and implant stability compared to an uncoated reference. The implants were coated using collagen-I with either two different concentrations of chondroitin sulfate (CS) or two differentially sulfated hyaluronans. Implant coatings were characterized biochemically and with atomic force microscopy. Histomorphometry was used to assess bone-implant contact (BIC) and bone-volume density (BVD) after 4 and 8 weeks of submerged healing in the maxilla of 20 minipigs. Further, implant stability was measured by resonance frequency analysis (RFA). Implants containing the lower CS concentration had significantly more BIC, compared to the uncoated reference at both times of interest. No significant increase was measured from week 4 to 8. Differences in BVD and RFA were statistically not significant. A higher concentration of CS and the application of sulfated hyaluronans showed no comparable increase in BIC. This study demonstrates a positive effect of a specific collagen-glycosaminoglycan combination on early bone formation in vivo.

Increased bone formation around coated implants.

AIM We hypothesized that coating threaded, sandblasted acid-etched titanium implants with collagen and chondroitin sulphate (CS) increases bone formation and implant stability, compared with uncoated controls. MATERIALS AND METHODS Three different implant surface conditions were applied: (1) sandblasted acid-etched (control), (2) collagen/chondroitin sulphate (low-dose--CS1), (3) collagen/chondroitin sulphate (high-dose--CS2). Sixty 9.5 mm experimental implants were placed in the mandible of 20 minipigs. Bone-implant contact (BIC) and relative peri-implant bone-volume density (rBVD--relation to bone-volume density of the host bone) were assessed after 1 and 2 months of submerged healing. Implant stability was measured by resonance frequency analysis (RFA). RESULTS After 1 month, coated implants had significantly more BIC compared with controls (CS1: 68%, p<0.0001, CS2: 63%, p=0.009, control: 52%). The rBVD was lower for all surface conditions, compared with the hostbone. After 2 months, BIC increased for all surfaces. No significant differences were measured (CS1: 71%, p=0.016, CS2: 68%, p=0.67, control: 63%). The rBVD was increased for coated implants. RFA values were 71-77 at implantation, 67-73 after 1 month and 74-75 after 2 months. Differences in rBVD and RFA were not statistically significant. CONCLUSIONS Data analysis suggests that collagen/CS has a positive influence on bone formation after 1 month of endosseous healing.

Functionalization of biomaterial surfaces using artificial extracellular matrices

Construction of biomaterials with the ability to guide cell function is a topic of high interest in biomaterial development. One approach is using components native to the ECM of the target tissue to generate in vitro a microenvironment that can also elicit specific responses in cells and tissues—an artificial ECM (aECM). The focus is on collagen as the basic material, which can be modified using a number of different glycoproteins, proteoglycans and glycosaminoglycans. Preparation, immobilization and the biochemical characteristics of such aECM are discussed, as well as the in vitro and in vivo response of cells and tissues, illustrating the potential of such matrices to direct cell fate.

Suitability of differently designed matrix-based implant surface coatings: an animal study on bone formation.

INTRODUCTION The aim of the present study was to assay how bone formation around dental implants is influenced by differently composed collagen matrices and RGD peptide as implant surface coatings compared to a sandblasted titanium surface. MATERIAL AND METHODS Five different implant surface coatings were designed: titanium (sandblasted), collagen type I, collagen type I&III, RGD-peptide, and mineralized collagen. Sixty experimental implants of a square-shaped design were inserted into the mandibles of 12 minipigs, 3 months following extraction of the premolar teeth. During the 6-month study period, sequential polyfluorochrome labeling was performed. After sacrifice, bone implant contact (BIC) was evaluated using histologic and histomorphometric methods. RESULTS New bone formation was observed against all implant surfaces. Polyfluorochrome labeling showed that bone growth started from the host bone in the majority of samples. The highest BIC was measured for collagen I and collagen I/III coated implants; however, significant differences between the coatings could not be found. CONCLUSION Osseointegration was achieved for all implant surfaces. Although a statistically significant increase in BIC could not be demonstrated for the experimental coatings after the 6 months study period, there was also no discernible detrimental effect of the coatings in comparison to the uncoated titanium surfaces.

Physicochemical and cell biological characterization of PMMA bone cements modified with additives to increase bioactivity

Polymethylmethacrylate (PMMA) bone cement is the most widely used material in surgery to fix joint replacements in the bone. In this study, we propose a new approach to generate bioactive PMMA surfaces directly at the site of implantation by adding the amphiphilic molecule phosphorylated 2-hydroxyethylmethacrylate (HEMA-P) to commercial PMMA bone cement, both with or without addition of 1-5% soluble calcium and carbonate salts. The setting behavior as well as the mechanical properties, the bonding quality at the metal/cement interface, mineral deposition, and cellular response for different cement modifications were investigated in vitro. The addition of HEMA-P resulted in entirely positive effects with respect to proliferation and differentiation of osteoblast-like cells (SaOs-2) and a very tight contact at the metal/cement interface. No detrimental changes of other properties were detected. The additional incorporation of salts provoked an increased deposition of calcium phosphate minerals but no further improvement in SaOs-2 cell differentiation. A significant decrease in polarization resistance for cements with high salt content (5%) was attributed to debonding between metal and cement. The results suggest an improved clinical performance of PMMA/HEMA-P composites, which might be further enhanced by small amounts of the soluble salts.

Collagen Type I Prevents Glyoxal-Induced Apoptosis in Osteoblastic Cells Cultured on Titanium Alloy

Advanced glycation end products (AGEs) irreversibly cross-link proteins with sugars and accumulate at a higher age and in diabetes, processes which can interfere with the integration of implants into the tissue. Glyoxal is a highly reactive glycating agent involved in the formation of AGEs and is known to induce apoptosis, as revealed by the upregulation of caspase-3 and fractin (caspase-3 being a key enzyme activated during the late stage of apoptosis and fractin being a caspase-cleaved actin fragment). In this study, we investigated the influence of collagen type I coating on the cytotoxic effect of glyoxal on rat calvarial osteoblastic cells and on human osteosarcoma cells (Saos-2) grown on titanium alloy, Ti6Al4V. Activation of caspase-3 and fractin was measured by counting immunohistochemically stained cells and by flow cytometry with propidium iodide (detection of the apoptosis indicating a sub-G1 peak). Our results showed an increased number of apoptotic osteoblasts after incubation with glyoxal on Ti6Al4V discs. However, the number of apoptotic cells on collagen-coated titanium was significantly smaller than on uncoated titanium after the same treatment. The present findings demonstrate that osteoblasts treated with glyoxal undergo apoptosis, whereas collagen type I coating of titanium alloys (used for implants) has an antiapoptotic function.

Glucuronic acid and phosphoserine act as mineralization mediators of collagen I based biomimetic substrates

Glucuronic acid (GlcA) and phosphoserine (pS) carrying acidic functional groups were used as model molecules for glycosaminoglycans and phosphoproteins, respectively to mimic effects of native biomolecules and influence the mineralization behaviour of collagen I. Collagen substrates modified with GlcA showed a stable interaction between GlcA and collagen fibrils. Substrates were mineralized using the electrochemically assisted deposition (ECAD) in a Ca2+/HxPO4(3−x) electrolyte at physiological pH and temperature. During mineralization of collagen–GlcA matrices, crystalline hydroxyapatite (HA) formed earlier with increasing GlcA content of the collagen matrix, while the addition of pS to the electrolyte succeeded in inhibiting the transformation of preformed amorphous calcium phosphate (ACP) to HA. The lower density of the resulting mineralization and the coalesced aggregates formed at a certain pS concentration suggest an interaction between calcium and the phosphate groups of pS involving the formation of complexes. Combining GlcA-modified collagen and pS-modified electrolyte showed dose-dependent cooperative effects.

Electrochemically Assisted Deposition of Thin CaP Coatings

Calcium phosphate (CaP) coatings have long been known to have a positive influence on implant integration into bone. Several methods for the formation of such coatings are known; this chapter focuses on the process of electrochemically assisted deposition (ECAD). With this highly reproducible biomimetic method, CaP coatings are generated on electrically conductive substrates in aqueous solutions. The fact that the process can take place at ambient temperature and neutral pH allows the deposition of coatings containing inorganic and especially organic components. Following a general description of the method and its basic principle, the influence of the deposition conditions on the coating properties is discussed in detail regarding a number of parameters. Among them are electrolyte composition, temperature, electrochemical mode used, and the effect of organic or inorganic additives. Variations of the said parameters can affect the composition of the phases deposited, the total coating mass, the mechanical characteristics, and a number of other properties. In addition to the physicochemical stability of ECAD coatings, another important aspect is the biological efficiency and compatibility of the coatings. In this context, cell biological investigations, animal studies, and experience from clinical applications are reviewed. The final aspect addressed in the chapter focuses on the characterization of ECAD-derived coatings in relation to the regulatory pathway and the prospects for industrial applications.