Tartrate-resistant acid phosphatase 5b (TRAP 5b) as a marker of osteoclast activity in the early phase after cementless total hip replacement.

BACKGROUND: After total hip replacement, increased bone metabolism is seen. A local periprosthetic osteopenia can be measured by dual-energy X-ray absorptiometry (DXA), but it is still unkown whether biochemical markers can be used to monitor the local remodeling at an earlier stage. PATIENTS AND METHODS: In this prospective study we compared the biochemical markers tartrate-resistant acid phosphatase 5b (TRAP 5b), bone ALP, osteocalcin and CrossLaps with periprosthetic DXA in 17 consecutive patients after uncemented total hip replacement. RESULTS: We found a highly significant early increase in TRAP 5b after 2 weeks and 6 weeks, which was followed by a densitometrically detectable decrease in bone mineral density after 26 weeks, especially in periprosthetic section Gruen zone 7. Bone ALP and osteocalcin levels as markers of osteoblast activity, and also Cross-Laps as a further marker of osteoclast activity, did not appear to allow any significant prediction of local bone remodeling. DISCUSSION: Our findings show that TRAP 5b is a sensitive parameter for monitoring of osteoclast activity after cementless total hip replacement, and may predict local osteopenia.

Measurement of varus angulation after femoral varus osteotomy in Legg-Calvé-Perthes disease.

Femoral varus osteotomy is an established therapy for Legg-Calvé-Perthes disease. The amount of surgical varus angulation is usually measured as the difference between the preoperative and the postoperative projected femur neck-shaft angle. We developed a more accurate measurement technique that considers changes of leg position and assessed the surgical varus angulation on two postoperative radiographs of each of 65 uniplanar femoral varus osteotomies. The mean difference between both surgical varus angulation measurements was 2.0 degrees +/- 1.4 degrees (range 0.0-5.0 degrees), the Pearson's correlation result was r = 0.96. The mean surgical varus angulation was 18.7 degrees +/- 7.3 degrees (range -2.5 to 43.5 degrees). Comparison with the preoperatively planned angulation showed a mean difference of 6.6 +/- 5.2 degrees (range 0.25-22.5 degrees).

Nanocrystalline hydroxyapatite and calcium sulphate as biodegradable composite carrier material for local delivery of antibiotics in bone infections.

The use of polymethylmetacrylate beads for local delivery of antibiotics requires a second surgical procedure for their removal and resorbable calcium sulphate exhibits cytotoxic effects. In this work, a bioresorbable composite of calcium sulphate and nanoparticulate hydroxyapatite (PerOssal was studied regarding its antibiotic release properties and biocompatibility. Material characteristics of plain PerOssal and pure calcium sulphate pellets were studied using scanning and electron microscopy and X-ray methods. Pellets were soaked with gentamicin and vancomycin, respectively. Release properties of both antibiotics from both materials were investigated over 10 days. Quantitative and qualitative cytotoxic assays were performed for biocompatibility testing. Specific surface was 106 m(2)/g for PerOssal and 2.2 m(2)/g for pure calcium sulphate. Almost complete elution of gentamicin was found for both carrier materials (94.7% for PerOssal vs. 95.8% for calcium sulphate) within 10 days, whereas vancomycin release was higher for PerOssal (96.3% vs. 74.8%). PerOssal showed higher initial and lower release after approximately 5 days compared to calcium sulphate. No significant in vitro cytotoxic differences were found between PerOssal and nontoxic cell culture medium. Calcium sulphate showed cytotoxic effects in two out of four tests. PerOssal exhibits excellent properties regarding resorption, biocompatibility, and antibiotic release.

The inflammatory responses to silk films in vitro and in vivo.

Silks have a long history of biomedical use as sutures. Silk can be purified, chemically modified to attach RGD sequences and processed into highly porous scaffolds for tissue engineering. We report biocompatibility studies of silk films (with or without covalently bound RGD) that were seeded with bone-marrow derived mesenchymal stem cells (MSC) and (a) cultured in vitro with human MSC or (b) seeded with autologous rat MSC and implanted in vivo. Controls for in vitro studies included tissue culture plastic (TCP; negative control), TCP with lipopolysaccharide (LPS) in the cell culture medium (positive control), and collagen films; controls for in vivo studies included collagen, PLA and TCP. After 9 h of culture, the expression of the pro-inflammatory Interleukin 1 beta (IL-1beta) and inflammatory cyclooxygenase 2 (COX-2) in human MSC were comparable for silk, collagen and TCP. After 30 and 96 h, gene expression of IL-1beta and COX-2 in MSC returned to the baseline (pre-seeding) levels. These data were corroborated by measuring IL-1beta and prostaglandin E2 levels in culture medium. The rate of cell proliferation was higher on silk films than either on collagen or TCP. In vivo, films made of silk, collagen or PLA were seeded with rat MSCs, implanted intramuscularly in rats and harvested after 6 weeks. Histological and immunohistochemical evaluation of silk explants revealed the presence of circumferentially oriented fibroblasts, few blood vessels, macrophages at the implant-host interface, and the absence of giant cells. Inflammatory tissue reaction was more conspicuous around collagen films and even more around PLA films when compared to silk. These data suggest that (a) purified degradable silk is biocompatible and (b) the in vitro cell culture model (hMSC seeded and cultured on biomaterial films) gave inflammatory responses that were comparable to those observed in vivo.

Engineering cartilage-like tissue using human mesenchymal stem cells and silk protein scaffolds.

Human mesenchymal stem cells (hMSC) derived from bone marrow aspirates can form the basis for the in vitro cultivation of autologous tissue grafts and help alleviate the problems of immunorejection and disease transmission associated with the use of allografts. We explored the utility of hMSC cultured on protein scaffolds for tissue engineering of cartilage. hMSC were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, and seeded on scaffolds. Four different scaffolds were tested, formed as a highly porous sponge made of: 1) collagen, 2) cross-linked collagen, 3) silk, and 4) RGD-coupled silk. Cell-seeded scaffolds were cultured for up to 4 weeks in either control medium (DMEM supplemented with 10% fetal bovine serum) or chondrogenic medium (control medium supplemented with chondrogenic factors). hMSC attachment, proliferation, and metabolic activity were markedly better on slowly degrading silk than on fast-degrading collagen scaffolds. In chondrogenic medium, hMSC formed cartilaginous tissues on all scaffolds, but the extent of chondrogenesis was substantially higher for hMSC cultured on silk as compared to collagen scaffolds. The deposition of glycosaminoglycan (GAG) and type II collagen and the expression of type II collagen mRNA were all higher for hMSC cultured on silk than on collagen scaffolds. Taken together, these results suggest that silk scaffolds are particularly suitable for tissue engineering of cartilage starting from hMSC, presumably due to their high porosity, slow biodegradation, and structural integrity.

Engineering bone-like tissue in vitro using human bone marrow stem cells and silk scaffolds.

Porous biodegradable silk scaffolds and human bone marrow derived mesenchymal stem cells (hMSCs) were used to engineer bone-like tissue in vitro. Two different scaffolds with the same microstructure were studied: collagen (to assess the effects of fast degradation) and silk with covalently bound RGD sequences (to assess the effects of enhanced cell attachment and slow degradation). The hMSCs were isolated, expanded in culture, characterized with respect to the expression of surface markers and ability for chondrogenic and osteogenic differentiation, seeded on scaffolds, and cultured for up to 4 weeks. Histological analysis and microcomputer tomography showed the development of up to 1.2-mm-long interconnected and organized bonelike trabeculae with cuboid cells on the silk-RGD scaffolds, features still present but to a lesser extent on silk scaffolds and absent on the collagen scaffolds. The X-ray diffraction pattern of the deposited bone corresponded to hydroxyapatite present in the native bone. Biochemical analysis showed increased mineralization on silk-RGD scaffolds compared with either silk or collagen scaffolds after 4 weeks. Expression of bone sialoprotein, osteopontin, and bone morphogenetic protein 2 was significantly higher for hMSCs cultured in osteogenic than control medium both after 2 and 4 weeks in culture. The results suggest that RGD-silk scaffolds are particularly suitable for autologous bone tissue engineering, presumably because of their stable macroporous structure, tailorable mechanical properties matching those of native bone, and slow degradation.

Bone tissue engineering using human mesenchymal stem cells: effects of scaffold material and medium flow.

We report studies of bone tissue engineering using human mesenchymal stem cells (MSCs), a protein substrate (film or scaffold; fast degrading unmodified collagen, or slowly degrading cross-linked collagen and silk), and a bioreactor (static culture, spinner flask, or perfused cartridge). MSCs were isolated from human bone marrow, characterized for the expression of cell surface markers and the ability to undergo chondrogenesis and osteogenesis in vitro, and cultured for 5 weeks. MSCs were positive for CD105/endoglin, and had a potential for chondrogenic and osteogenic differentiation. In static culture, calcium deposition was similar for MSC grown on collagen scaffolds and films. Under medium flow, MSC on collagen scaffolds deposited more calcium and had a higher alcaline phosphatase (AP) activity than MSC on collagen films. The amounts of DNA were markedly higher in constructs based on slowly degrading (modified collagen and silk) scaffolds than on fast degrading (unmodified collagen) scaffolds. In spinner flasks, medium flow around constructs resulted in the formation of bone rods within the peripheral region, that were interconnected and perpendicular to the construct surface, whereas in perfused constructs, individual bone rods oriented in the direction of fluid flow formed throughout the construct volume. These results suggest that osteogenesis in cultured MSC can be modulated by scaffold properties and flow environment.