Chondrocyte behaviour within different types of collagen gel in vitro.

In cartilage repair experiments chondrocytes are transplanted into osteochondral defects. Biological substances are used as cell vehicles and are likely to play an important role in the outcome of these studies. Collagen gel is formed by polymerization of type I collagen and is used in plastic surgery and for three-dimensional culture systems. To test collagen gel as a potential vehicle for transplantation, we evaluated chondrocyte behaviour in vitro in different collagen gels. Collagen type I was extracted and purified from rat tail tendon and fetal calf skin and compared with commercially available collagen type I. After suspension of bovine chondrocytes, five different collagen gels were cultured for 14 days and evaluated by light and electron microscopy. Cells proliferated within all gels and synthesized proteoglycans as assessed by 35S incorporation; 40-90% of cells maintained a chondrocyte-like morphology after 1 week in culture depending on the type of collagen gel. Synthetic and secretory activity was confirmed by electron microscopy. Based on these results, calf skin collagen is recommended for culturing chondrocytes for implantation.

Calcitonin gene-related peptide, substance P and GAP-43/B-50 immunoreactivity in the normal and arthrotic knee joint of the mouse.

The aim of this study was to describe the normal distribution of calcitonin gene-related peptide (CGRP) and substance P (SP) containing fibres in the knee joint of the mouse and to obtain insight into the changes in innervation associated with degenerative processes in the joint. Arthrosis was induced by a single subpatellar intra-articular injection of bacterial collagenase. After decalcification in EDTA solutions, the CGRP and SP fibres were visualized by peroxidase-antiperoxidase pre-embedding immunocytochemistry for light microscopy. Control experiments on the mouse brain as a reference for the effect of EDTA on the immunostaining showed that the decalcification procedure with EDTA had not impaired the immunostaining. A rich innervation of thin varicose CGRP and SP immunoreactive fibres was found in most peri- and intra-articular tissue components. The periosteum, synovial tissues, the joint capsule and the intra-articular fat tissues were richly innervated. Less intense innervations were also found in the subchondral bone plates of the tibio-femoral joint and of the patella. Fibres were also found in the soft tissues between the patellar tendon and the femoral groove. No differences could be found between the location of CGRP and SP fibres with respect to the localization in the joint, but generally more CGRP fibres were found. The collagenase-induced osteoarthrosis was characterized by sclerosis of the subchondral bone, patellar dislocation, osteophyte formation, synovial proliferation and by severe cartilage abrasion, particularly on the medial side of the femoro-tibial joint. The overall distribution of CGRP and SP fibres was the same as in the control joints. However, major differences were found in all studied joints at specific locations around the cruciate ligaments, in the synovium around the patella, in the soft tissues lateral of the patella and in plica tissue between the patella and femoral groove. The CGRP and SP innervation was no longer detectable by immunolabelling with the antibodies. With a polyclonal antibody to the growth associated protein GAP-43/B-50, signs of degenerated axonal profiles were observed in these locations. At other peripheral locations, such as the muscles, the GAP-43/B-50 distribution was normal. In conclusion, the present study provides detailed information on the localization of CGRP and SP fibres, which may be involved in pain perception. Knowledge of the changes that occur during arthrosis may give more insight into the clinical symptoms.

The repeated sampling bone chamber: a new permanent titanium implant to study bone grafts in the goat.

We developed a repeated sampling bone chamber (RSBC) and tested its suitability for studying various aspects of the bone allograft incorporation process under reproducible nonload-bearing experimental conditions in a large vertebrate. Our chamber is made of commercially pure titanium and is designed to allow bone or tissue ingrowth into a removable hollow inner core. Three chambers per animal were randomly implanted in the tibias of 10 goats and were harvested every 8 weeks. In experiment 1, two chambers were filled with a fresh-frozen structural allograft or a chip allograft, and one was left empty. In experiment 2, all chambers were left empty to measure intra- and interanimal variation. The results were evaluated by histomorphometry. Clinical results of four growth factor experiments also are presented. Using this model, we conducted 60 harvest operations (median, 4/animal; range, 2 to 8). In experiment 1, more soft tissue ingrowth and osteoclasts were measured in the chambers with allograft (P < 0.005 and P < 0.03 respectively). Bone ingrowth was scant, with no significant differences between chip graft, structural graft, and empty control chamber. Thus, the bone graft did not show any osteoinductive or osteoconductive properties. Experiment 2 indicated consistent tissue ingrowth, with greater interanimal variation than variations among the chambers in any goat. Our method forms a means of studying gradual tissue and bone ingrowth into bone grafts. The inherent low amount of bone ingrowth makes this model suitable for studying bone-inductive substances. Repeated sampling in the same animals lowered the intersample variability and reduced the number of animals that were required.

Impacted graft incorporation after cemented acetabular revision. Histological evaluation in 8 patients.

We took core biopsies from the acetabulum in 8 patients (at reoperation) after a previous revision with impacted cancellous allograft chips in combination with cement. Except for one biopsy specimen, the graft showed different stages of incorporation. In the specimens taken at 4 months, revascularization of the graft was found. Osteoclasts had removed parts of the graft, while woven bone had formed on the remnants of the graft and in the stroma that was invading the graft. Subsequent specimens showed that this mixture of graft and new bone was in due time remodeled into a normal trabecular bony structure with viable bone marrow that contained little or no remnants of the original graft. The graft-cement interface was present in 4 biopsies taken at 1, 22, 28, and 72 months. The specimen obtained 28 months after revision showed vital bone locally in direct contact with the cement layer; however, a soft tissue interface predominated.

Acetabular reconstruction with impacted morselized cancellous allografts in cemented hip arthroplasty: a histological and biomechanical study on the goat.

Bone defects in total hip arthroplasty revision surgery can be restored with different types of bone graft. The use of impacted morselized allograft chips in combination with cement is the treatment of our choice. To establish the incorporation capacity of the grafts and mechanical stability of the implant, an animal model in the goat was developed. An acetabular defect was created and restored with morselized grafts and a cemented cup. Postoperative performance of the reconstruction was followed both histologically and biomechanically. Histology showed that consolidation of the graft with the host bone bed had occurred within 3 weeks. In the following period a front of vascular sprouts infiltrated the graft. Graft resorption, woven bone deposition, and subsequent remodeling resulted in a new trabecular structure. This structure contained only scarce remnants of the original dead graft material. At the graft-cement interface, graft resorption and new bone formation had resulted in areas of direct vital bone-cement contact. Locally, a soft tissue interface was present. After longer follow-up periods, progressive interface formation and loosening of the cups were found in most animals. Mechanical testing showed that the stability of the reconstruction increased during the first 12 postoperative weeks. Thereafter, the stability decreased, probably by soft-tissue interface formation at the graft cement interface. We conclude that cemented morselized allografts have a high capacity to incorporate. Initial cup stability is adequate to provoke graft incorporation with decreasing stability after the incorporation process has been completed.

The effect of biphosphonate on induced heterotopic bone.

HEBP (1-hydroxy, ethylidene-1, 1-biphosphonate) inhibited mineralization and was observed in matrix-induced heterotopic bone in rabbits. In one group of rabbits, HEBP was administered continuously until sacrifice 20 weeks after the operation. Another group of animals received HEBP for the first four weeks only. The effect of HEBP on de novo bone formation was determined by histologic and biochemical analyses. Implant alkaline and acid phosphatase levels and implant calcium and phosphate contents were measured. The implants of HEBP-treated animals showed diminished implant resorption and, at the same time, formation of atypical osteoid tissue. Quantitative measurements revealed a decrease of acid phosphatase activity, whereas implant alkaline phosphatase activity was unaffected. The mineralization, as depicted by the implant calcium and phosphate content, was almost completely inhibited during HEBP-administration. These effects were completely reversible after the withdrawal of the drug. Remineralization began directly after discontinuation, and recovered only 12 weeks later. The results of this study confirm reports that HEBP cannot prevent the formation of heterotopic ossification. The only effect would be a delay of mineralization during its administration.

Viability of the acetabular bone bed at revision surgery following cemented primary arthroplasty.

Loosening of total hip replacements is often associated with severe loss of periprosthetic bone. The notion exists that the remaining bone is sclerotic, avascular, and displays little osteogenic activity, and that it therefore potentially compromises the revitalization of bone grafts used to restore bony defects. To verify this opinion we studied the bone characteristics in acetabular bone biopsies taken at primary total hip arthroplasty (PTH) and revision total hip arthroplasty (RTH) for a cemented PTH. In 6 PTH patients and in 10 RTH patients, acetabular bone biopsies were taken from the roof, the center, and the lower rim of each acetabulum. Specimens were evaluated by light microscopy and histomorphometrically measured for specimen size, bone area, perimeter, active osteoid perimeter, number of vessels, and osteoclasts. The vascularity and vitality appeared to be comparable in the RTH and PTH bone biopsies. However, the trabecular organization of the RTH bone differed from that of the PTH biopsies. In the PTH biopsies, the trabeculae were running perpendicular to the subchondral bone layer, whereas in the RTH biopsies the layers of bone were oriented parallel to the implant surface. There was abundant remodeling activity in the RTH bone, with large quantities of active osteoid and osteoclasts. These histologic parameters differed, but not statistically significant, from the PTH biopsies. In conclusion, we found that at revision, the acetabular bone was viable with sufficient vascularity and remodeling activity to provide an acceptable recipient host bone bed for revision surgery combined with bone grafting.

Culture of chondrocytes in alginate and collagen carrier gels.

In this in vitro study, we compared the potential of collagen and alginate gels as carriers for chondrocyte transplantation and we studied the influence of demineralized bone matrix (DBM) on chondrocytes in the gels. Chondrocytes were assessed for cell viability, phenotype (histology), proliferation rate and sulfate incorporation. Collagen gels showed a significant increase in cell numbers, but the chondrocytes dedifferentiated into fibroblast-like cells from day 6 onwards. In alginate gels, initial cell loss was found, but the cells maintained their typical chondrocyte phenotype. Although the total quantity of proteoglycans initially synthesized per cell in collagen gel was significantly higher, expressed per cell, the quantity in alginate gel eventually surpassed collagen. No effects of culturing chondrocytes in combination with DBM could be demonstrated on cell proliferation and sulfate incorporation. The collagen and alginate gels have different advantages as carriers for chondrocyte transplantation. The high proliferation rate of chondrocytes in collagen gel may be an advantage, but the preservation of the chondrocyte phenotype and the gradually increasing proteoglycan synthesis in alginate gel is a promising method for creating a hyaline cartilage implant in vitro.