Effects of copper-impregnated collagen implants on local pro- and anti-inflammatory and regenerative tissue reactions following implantation in rats.

Combining collagen, an established regenerative biomaterial, and copper (Cu) with its known antibacterial and angiogenic effects could improve wound healing. However, Cu is also cytotoxic. Thus, this study aimed at examining the tissue reactions after simultaneous intramuscular implantation of collagen discs either without Cu (controls) or impregnated in 2, 20, or 200 mmol/L Cu acetate in 24 rats. After 7, 14, and 56 days, implants with peri-implant tissue were retrieved from 8 rats/day for immunohistochemical detection of CD68+ monocytes/macrophages and CD163+ macrophages, MHC-II+ cells, T lymphocytes and nestin as tissue regeneration marker. CD68+ monocytes/macrophages around implants increased with Cu amount but decreased over time except for the highest Cu amount, while CD163+ macrophages increased over time around and within implants. MHC-II+ cells were similar to CD68+ monocytes/macrophages. T lymphocyte numbers around implants were higher for Cu-impregnated samples vs. controls on day 7 and highest on day 14, but declined afterwards. Nestin expression around and within implants was largely unaffected by Cu. In conclusion, pro-inflammatory reactions around implants were dose-dependently influenced by Cu but mostly decreased over time, while Cu did not negatively affect anti-inflammatory and regenerative reactions. These results suggest that Cu-impregnated collagen could be beneficial in wound treatment.

Antimicrobial and bone-forming activity of a copper coated implant in a rabbit model.

Current strategies in implant technology are directed to generate bioactive implants that are capable to activate the regenerative potential of the surrounding tissue. On the other hand, implant-related infections are a common problem in orthopaedic trauma patients. To meet both challenges, i.e. to generate a bone implant with regenerative and antimicrobial characteristics, we tested the use of copper coated nails for surgical fixation in a rabbit model. Copper acetate was galvanically deposited with a copper load of 1 µg/mm2 onto a porous oxide layer of Ti6Al4V nails, which were used for the fixation of a tibia fracture, inoculated with bacteria. After implantation of the nail the concentration of copper ions did not increase in blood which indicates that copper released from the implant was locally restricted to the fracture site. After four weeks, analyses of the extracted implants revealed a distinct antimicrobial effect of copper, because copper completely prevented both a weak adhesion and firm attachment of biofilm-forming bacteria on the titanium implant. To evaluate fracture healing, radiographic examination demonstrated an increased callus index in animals with copper coated nails. This result indicates a stimulated bone formation by releasing copper ions. We conclude that the use of implants with a defined load of copper ions enables both prevention of bacterial infection and the stimulation of regenerative processes.

Corrigendum to 'In vivo evaluation of copper release and acute local tissue reactions after implantation of copper-coated titanium implants in rats'.

In the "Materials and methods" chapter of the article given above, in section 2.1 titled "Implant samples and preparation" on page 2 (top paragraph of the right column), an incorrect value of 1 µg mm-2 for the average Cu load of the surface was inadvertently specified. This was due to an accidental mix-up of electronic data sets from examination of different implant series. The correct value is 0.2 µg mm-2. No other data given in the article are affected. The authors kindly apologize for this mistake.

A dual function of copper in designing regenerative implants.

The supply of titanium implants which are widely used in orthopaedics with both regenerative and anti-microbial properties will achieve a great progress in bone regeneration. We asked, whether by appropriate concentrations of copper ions it will be possible both to inhibit growth of bacteria and stimulate biological responses in mesenchymal stem cells (MSC). Using titanium material which released galvanically deposited copper at concentrations from 0.3 to 1.75 mM, growth of planktonic Staphylococcus aureus was blocked and more importantly adherent bacteria were cleared from the material surface within 24 h. To test biological responses of human bone marrow derived MSC due to copper ions, we found that copper stimulated the proliferation of MSC in a narrow concentration range around 0.1 mM. Similar copper concentrations enhanced osteogenic differentiation of MSC when cells were cultured in osteogenic differentiation medium. We observed increased activity of alkaline phosphatase (ALP), higher expression of collagen I, osteoprotegerin, osteopontin and finally mineralization of the cells. We conclude that titanium implants that release copper ions can be effective against bacterial infections at higher concentrations of copper near the implant surface and can promote bone regeneration when its concentration becomes lower due to diffusion.

Endothelial cells stimulate osteogenic differentiation of mesenchymal stem cells on calcium phosphate scaffolds.

The interaction of mesenchymal stem cells (MSCs) with endothelium in vivo is significant for regenerative processes in organisms. To design concepts for tissue engineering for bone regeneration based on this interaction, the osteogenic differentiation of human bone marrow-derived MSCs in a co-culture with human dermal microvascular endothelial cells (HDMECs) was studied. The experiments were focussed on the regulation of MSCs in a co-culture with HDMECs on different calcium phosphate scaffolds. Alkaline phosphatase (ALP) activity and mRNA expression of various osteogenic markers increased significantly when cells were co-cultured on materials with calcium phosphate scaffolds compared to tissue culture polystyrene or when MSCs were cultured alone. In addition, it was observed that the expression of osteopontin and osteocalcin was highly sensitive to the substrate for cell adhesion. Whereas these late osteogenic markers were down-regulated in co-cultures on polystyrene, they were up-regulated on calcium phosphate and moreover, were differentially expressed on the three calcium phosphate scaffolds tested. To enhance the osteogenic differentiation of MSCs in a co-culture, direct cell-cell interactions were required. Concerning molecular mechanisms in the interactions between both cell types, it was found that connexin 43 was expressed in contact sites and more apparently, endothelial cells grew over the MSCs, which facilitated direct cellular interactions mediated by various adhesion receptors. This study revealed significant findings for the design of implant materials suitable for regeneration of bone by stimulating the functional interaction of MSCs with endothelial cells.

Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro.

Due to the weak regeneration potential of cartilage, there is a high clinical incidence of articular joint disease, leading to a strong demand for cartilaginous tissue surrogates. The aim of this study was to evaluate a gelatin-based hydrogel for its suitability to support chondrogenic differentiation of human mesenchymal stem cells. Gelatin-based hydrogels are biodegradable, show high biocompatibility, and offer possibilities to introduce functional groups and/or ligands. In order to prove their chondrogenesis-supporting potential, a hydrogel film was developed and compared with standard cell culture polystyrene regarding the differentiation behavior of human mesenchymal stem cells. Cellular basis for this study were human adipose tissue-derived mesenchymal stem cells, which exhibit differentiation potential along the adipogenic, osteogenic and chondrogenic lineage. The results obtained show a promotive effect of gelatin-based hydrogels on chondrogenic differentiation of mesenchymal stem cells in vitro and therefore encourage subsequent in vivo studies.

In vivo evaluation of copper release and acute local tissue reactions after implantation of copper-coated titanium implants in rats.

Copper (Cu) based coatings can reduce infections for titanium (Ti) implants. However, Cu is also cytotoxic. To examine the balance of antibacterial versus adverse tissue effects, this study aimed at evaluating a Cu coating regarding in vivo Cu release and local inflammatory reactions for 72 h. TiAl6V4 plates received either plasma electrolytic oxidation only (Ti), or an additional galvanic Cu deposition (Ti-Cu). No Staphylococcus aureus were found in vitro on Ti-Cu after 24 h. Following simultaneous intramuscular implantation of two Ti and two Ti-Cu plates into nine rats, serum Cu was elevated until 48 h and residual Cu on explanted samples reduced accordingly after 48 h. Total and tissue macrophages around implants increased until 72 h for both series, and were increased for Ti-Cu. As numbers of total and tissue macrophages were comparable, macrophages were probably tissue-derived. MHC-class-II-positive cells increased for Ti-Cu only. T-lymphocytes had considerably lower numbers than macrophages, did not increase or differ between both series, and thus had minor importance. Tissue reactions increased beyond Cu release, indicating effects of either surface-bound Cu or more likely the implants themselves. Altogether, Ti-Cu samples possessed antibacterial effectiveness in vitro, released measurable Cu amounts in vivo and caused a moderately increased local inflammatory response, demonstrating anti-infective potential of Cu coatings.

Morphometric immunohistochemical examination of the inflammatory tissue reaction after implantation of calcium phosphate-coated titanium plates in rats.

Calcium phosphate (CaP) preparations are established coatings for titanium-based medical implants used for bone reconstruction. However, biodegradation of the coating can result in microparticles that subsequently cause inflammatory reactions. The present study was therefore aimed at investigating the inflammatory response to two series of CaP-coated titanium plates: Ti-brushite (Ti-B) and Ti-hydroxyapatite (Ti-H) implants. Fifteen male LEW.1A rats received one plate of each series and a pellet (5 x 2 mm) of sol-gel derived silica/CaP (SCP implants) implanted into the back musculature. After 7, 14 and 28 days, five rats were killed and the implants were removed with the surrounding tissue. Quantitative immunohistochemistry was performed on frozen sections. Total monocytes/macrophages, tissue macrophages, T-cells, MHC-class-II-positive cells and proliferating cells were counted. For the Ti-B implants, the number of monocytes/macrophages remained constant while the other cell populations increased. In contrast, for the Ti-H implants the number of monocytes/macrophages decreased while the other cell populations remained constant. The SCP implants demonstrated degradation and scattering into smaller particles with an increase for all cell populations except the proliferating cells. Human mesenchymal stem cells demonstrated adherence and a flat morphology on Ti-B and Ti-H implants and no remarkable difference between both implants. Taken together, the in vivo data demonstrate that the short-term inflammatory response against a hydroxyapatite coating is lower in comparison to a brushite coating, and that the morphology of cells growing in vitro is similar on both layers.

Biocompatibility studies of endothelial cells on a novel calcium phosphate/SiO2-xerogel composite for bone tissue engineering.

The bone biomaterial BONITmatrix, a nanoporous, granular scaffold composed of hydroxylapatite, calcium phosphate and SiO2, linked by a dense collagen mesh, was tested for its biocompatibility using endothelial cells (EC) in the form of macrovascular HUVEC, microvascular HDMEC and the endothelial cell line ISOHAS-1. Cells were examined for their adherence and growth on the biomaterial and this was followed by confocal laser scanning microscopy after vital staining or immunocytochemical reactions, as well as by scanning electron microscopy. Macro- and microvascular ECs predominantly spread on BONITmatrix-collagen mesh-covered surfaces and fibres and maintained their typical morphology. As ECs in vivo must build up a functional vasculature, the seeded cells were further tested for proinflammatory expression markers and cytokine expression after lipopolysaccharide stimulation. Protein-coating studies revealed that BONITmatrix-collagen scaffolds needed human blood serum coating to successfully support the growth of ECs. All cells expressed endothelium-specific surface marker proteins such as PECAM-1, VE-cadherin and vWF. The in vitro data support recent in vivo studies and indicate that this calcium phosphate/SiO2-xerogel composite could be a useful scaffold material for tissue engineering.

Calcium phosphate surfaces promote osteogenic differentiation of mesenchymal stem cells.

Although studies in vivo revealed promising results in bone regeneration after implantation of scaffolds together with osteogenic progenitor cells, basic questions remain how material surfaces control the biology of mesenchymal stem cells (MSC). We used human MSC derived from bone marrow and studied the osteogenic differentiation on calcium phosphate surfaces. In osteogenic differentiation medium MSC differentiated to osteoblasts on hydroxyapatite and BONITmatrix, a degradable xerogel composite, within 14 days. Cells revealed a higher alkaline phosphatase (ALP) activity and increased RNA expression of collagen I and osteocalcin using real-time RTPCR compared with cells on tissue culture plastic. To test whether material surface characteristics alone are able to stimulate osteogenic differentiation, MSC were cultured on the materials in expansion medium without soluble additives for osteogenic differentiation. Indeed, cells on calcium phosphate without osteogenic differentiation additives developed to osteoblasts as shown by increased ALP activity and expression of osteogenic genes, which was not the case on tissue culture plastic. Because we reasoned that the stimulating effect on osteogenesis by calcium phosphate surfaces depends on an altered cell-extracellular matrix interaction we studied the dynamic behaviour of focal adhesions using cells transfected with GFP labelled vinculin. On BONITmatrix, an increased mobility of focal adhesions was observed compared with cells on tissue culture plastic. In conclusion, calcium phosphate surfaces are able to drive MSC to osteoblasts in the absence of osteogenic differentiation supplements in the medium. An altered dynamic behaviour of focal adhesions on calcium phosphate surfaces might be involved in the molecular mechanisms which promote osteogenic differentiation.

Control of focal adhesion dynamics by material surface characteristics.

The mechanisms of cell adhesion to the extracellular matrix (ECM) which are of fundamental importance for function, survival, and growth of cells involve the formation of focal adhesions to facilitate integrin signaling. Recently, it became evident that focal adhesions are not stable but move to enable cell migration and ECM formation. We examined the number, size, and dynamic behavior of focal adhesions in living MG-63 osteoblastic cells, which were cultured on titanium surfaces with different roughnesses and on stainless steel (SS). As a marker for focal adhesions we used GFP-tagged vinculin, a cytoskeletal protein. Focal adhesions were smaller on titanium and on SS than on collagen-coated glass coverslips. The corundum-blasted rough surface of titanium induced the smallest adhesions. On all the surfaces that we have tested, we observed a mobility of focal adhesions. On collagen-coated coverslips focal adhesions moved with a speed of 60 nm/min. The speed was reduced on titanium and still more restricted on SS. The topography did not affect the mobility of focal adhesions. We conclude that on the material surfaces that we have studied a reduced mobility of focal adhesions may strengthen the linkages between cell and ECM but impair the ability to dynamically organize and remodel the ECM. The results may have a great impact in the functional evaluation of tailored biomaterial surfaces for the application in tissue engineering.

Cellular investigations on electrochemically deposited calcium phosphate composites.

Electrochemically deposited calcium phosphate (CaP) coatings are fast resorbable and existent only during the first period of osseointegration. In the present study, composite coatings with varying solubility (hydroxyapatite (HA), brushite with less HA and monetite (M) with less HA) were prepared and the influence of the degradation and the reprecipitation of CaP on osteoblastic cells were investigated. On the brushite composite coating a new precipitated, finely structured CaP phase was observed during immersion in cell culture medium with or without osteoblastic cells. The surface morphology of monetite and HA coatings were entirely unmodified under the same conditions. So it could be assumed that electrochemically deposited brushite with less HA acts as a precursor for new precipitated CaP. On this surface osteoblastic cells revealed a well-spread morphology with pronounced actin cytoskeleton and demonstrated good proliferation behaviour. Thus we suggest that brushite seems to be especially suitable for coating of implants as a matrix for nucleation and growth of new bone.