Expression of vascular endothelial growth factor on chondrocytes increases with osteoarthritis - an animal experimental investigation.

OBJECTIVE: To evaluate the expression of VEGF by chondrocytes of hyaline cartilage during the course of osteoarthritis (OA). METHODS: In 12 white New Zealand rabbits the anterior cruciate ligament (ACL) was resected to create an anterior instability of the knee. In 12 control rabbits only a sham operation without resection of the ACL was done. Four animals of each group were killed at 3, 6, and 12 weeks. The load bearing area was evaluated histologically according to Mankin and by immunostaining for VEGF. RESULTS: In the experimental group, histological grades of OA showed a positive linear correlation with the time after surgery. Immunostaining showed an increased expression of VEGF in the control group after 3 weeks, which dropped to normal after 6 weeks. There was no difference in the progression of OA between control and experimental groups after 3 weeks, but a significant difference was seen after 6 (p=0,01) and 12 (p=0,05) weeks. A significant positive correlation between VEGF expression and the histological grade of OA was found (r = 0.767; p<0.01). CONCLUSIONS: An increase of VEGF expressing chondrocytes occurs during time course of OA.

Influence of hepatocyte growth factor on autologous osteochondral transplants in an animal model.

PURPOSE: Several studies have investigated the influence of different growth factors on hyaline cartilage regeneration. In a rabbit model, hepatocyte growth factor (HGF) was proven to increase the amount of hyaline-like chondrocytes in a mixed fibro-cartilaginous regenerate of small defects. The aim of the current study was to evaluate whether intra-articular administration of HGF influences the ingrowth of osteochondral grafts in a sheep model. TYPE OF STUDY: Animal experiment. METHODS: Both knee joints of eight sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the opposite condyle of the same joint. The sheep were divided into two groups of four sheep, resulting in 16 grafts per group. In one group, HGF was administered by bilateral intra-articular injections given three times a week for 4 weeks. The control group received isotonic sodium chloride injections. The animals were killed after 3 months. RESULTS: Histological evaluation showed a complete ingrowth of the osseous part of the osteochondral grafts. A healing or ingrowth at the level of the cartilage could not be observed. Histological evaluation of the transplanted grafts according to the modified Mankin score revealed less degeneration in the cartilage of the HGF group, as compared to the control group. In the HGF group, less cloning of chondrocytes and less irregularities of the articular surface were observed. Importantly, no deleterious effects, such as osteophyte formation, cartilage thickening or synovial proliferation, were found. CONCLUSION: HGF positively influenced the cellularity of the transplanted osteochondral graft, but could not diminish the fissures in the marginal zone of the grafts. CLINICAL RELEVANCE: Marginal zone fissures and degeneration in the absence of HGF may undermine long-term results of autologous osteochondral grafts.

Mineralization at the interface of implants.

Osseointegration of implants is crucial for the long-term success of oral implants. Mineralization of the bone's extracellular matrix as the ultimate step of a mature bone formation is closely related to implant osseointegration. Osteogenesis at oral implants is a complex process, driven by cellular and acellular phenomena. The biological process of the maintenance and emergence of minerals in the vicinity of oral implants is influenced to a great extent by biophysical parameters. Implant-related structural and functional factors, as well as patient-specific factors, govern the features of osteogenesis. To understand the influence of these factors in peri-implant bone mineralization, it is important to consider the basic biological processes. Biological and crystallographic investigations have to be applied to evaluate mineralization at implant surfaces at the different hierarchical levels of analysis. This review gives insight into the complex theme of mineral formation around implants. Special focus is given to new developments in implant design and loading protocols aimed at accelerating osseointegration of dental implants.

Expression of the matrix receptor CD44v5 on chondrocytes changes with osteoarthritis: an experimental investigation in the rabbit.

OBJECTIVE: To evaluate the expression of CD44v5 on chondrocytes of hyaline cartilage during the course of osteoarthritis (OA). METHODS: In 12 white New Zealand rabbits the anterior cruciate ligament (ACL) was resected to create an anterior instability of the knee. In 12 control rabbits only a sham operation without resection of the ACL was done. Four animals of each group were killed at 3, 6, and 12 weeks. The loadbearing area was evaluated histologically according to Mankin and by immunostaining for CD44v5. RESULTS: In the trial group, histological grades of OA showed a positive linear correlation with the time after surgery. Immunostaining showed an increased expression of CD44v5 in the control group after 3 and 6 weeks, which dropped to normal after 12 weeks. There was no difference between control and trial groups after 3 and 6 weeks, but a difference was seen after 12 weeks. A significant positive correlation between CD44v5 expression and the histological grade of OA was found (r = 0.314). CONCLUSIONS: An in vivo increase of expression of the hyaluronan receptor CD44v5 occurs during the course of OA. Further studies are needed to evaluate whether this pattern applies to man and whether new treatment approaches might evolve from this knowledge.

Hyaline cartilage degenerates after autologous osteochondral transplantation.

OBJECTIVES: Autologous osteochondral grafting is a well-established clinical procedure to treat focal cartilage defects in patients, although basic research on this topic remains sparse. The aim of the current study was to evaluate (1) histological changes of transplanted hyaline cartilage of osteochondral grafts and (2) the tissue that connects the transplanted cartilage with the adjacent cartilage in a sheep model. METHOD: Both knee joints of four sheep were opened surgically and osteochondral grafts were harvested and simultaneously transplanted to the contralateral femoral condyle. The animals were sacrificed after three months and the received knee joints were evaluated histologically. RESULTS: Histological evaluation showed a complete ingrowth of the osseous part of the osteochondral grafts. A healing or ingrowth at the level of the cartilage could not be observed. Histological evaluation of the transplanted grafts according to Mankin revealed significantly more and more severe signs of degeneration than the adjacent cartilage, such as cloning of chondrocytes and irregularities of the articular surface. CONCLUSION: We found no connecting tissue between the transplanted and the adjacent cartilage and histological signs of degeneration of the transplanted hyaline cartilage. In the light of these findings, long-term results of autologous osteochondral grafts in human beings have to be followed critically.

Neuronal regeneration after application of radiofrequency energy to collagenous tissue is affected by limb immobilization: an in vivo animal study.

Despite widespread use of radiofrequency (RF)-shrinkage, there have been no studies on the influence of RF-energy on neural elements of collagenous tissue. The purpose of this study was to examine the effect of RF-shrinkage on neural structures of capsuloligamentous tissue and the recovery of neural elements under different postoperative treatment protocols. One patellar tendon of 46 New-Zealand-White rabbits was shrunk. Six rabbits were sacrificed immediately postoperative. Twenty rabbits were not immobilized, 10 were immobilized for 3 and 10 were immobilized for 6 weeks. A monoclonal antibody, specific against a neurofilament protein, was used to detect nerves and neural structures. Staining pattern of nerve fibres was significantly altered immediately postoperative. After 3 weeks the number of nerve fibres and bundles decreased significantly in immobilized and non-immobilized limbs. The loss of nerve fibres was significantly less in immobilized limbs. At 6 weeks the number of neural elements in immobilized limbs increased to the level of untreated control tissue. In non-immobilized limbs we found no recovery of neural elements 9 weeks postoperatively. At this time the number of nerve fibres and bundles was still significantly less compared to the untreated control limbs. RF-shrinkage causes significant alteration of neural elements. Under immobilization nerve fibres and bundles reach the level of normal untreated tissue. Careful rehabilitation is important after RF-shrinkage. Not only for biomechanical reasons, but also to allow the neural elements to recover, thermally modified tissue should be protected from normal physiologic loads.

Immobilisation after radiofrequency-induced shrinkage of tendon. A histological study in rabbits.

Despite widespread use of radiofrequency (RF) shrinkage, there have been no animal studies on the effects of post-operative immobilisation on the histological properties of the shrunken tissue. We have therefore examined the role of post-operative immobilisation after RF shrinkage with special emphasis on the histological properties of collagenous tissue. One patellar tendon of 66 New Zealand White rabbits was shrunk. Six rabbits were killed immediately after the operation. Twenty rabbits were not immobilised, 20 were immobilised for three weeks and 20 for six weeks. Fibroblasts, collagen and vascular quality and density were evaluated on sections, stained by haematoxylin and eosin. Nine weeks after operation the histological properties were inferior to those of the contralateral control tendons. Shrunk tendons did not return to normal at any time after operation irrespective of whether the animals had been immobilised or not. All the parameters improved significantly between zero and three weeks after operation. Immobilised tendons tended to have a better and faster recovery. Careful rehabilitation is imperative after RF shrinkage. Immobilisation aids recovery of the histological properties. Our findings in this animal model support a period of immobilisation of more than three weeks.

Vitamin E reduces platelet adhesion to human endothelial cells in vitro.

Although it has been reported that vitamin E (alpha-tocopherol) can reduce platelet adhesiveness and aggregation in vivo, the mechanism is still unknown. Therefore, the aim of the present study was to determine whether incubations of platelet-rich plasma (PRP) with vitamin E influence platelet adhesion to cultured endothelial cells. To exclude blood plasma involvement, also washed platelets were pretreated with alpha-tocopherol. Vitamin E (0.5-1.0 mM) was added to PRP or washed platelets. Endothelial cells in monolayer were incubated with thrombin-activated platelets (1 or 2 U/ml). After 1 hr of incubation, non-adhered platelets were removed and counted. Treating of PRP with alpha-tocopherol inhibited platelet adhesion to endothelial cell monolayer. This effect was dose dependent on concentrations of alpha-tocopherol and thrombin. In our experiments PRP was treated with alpha-tocopherol and endothelial cell monolayer was used as test surface. These findings agree with previous observations on the adhesivity of platelets to synthetic surfaces after dietary vitamin E in healthy volunteers. When washed platelets were incubated with alpha-tocopherol, no significant reduction of adhesion was detectable. As preincubation of washed platelets with alpha-tocopherol does not inhibit platelet adhesion, it may be supposed that the effect of vitamin E does not occur in a directly cellular mechanism. The data suggest that alpha-tocopherol may reduce platelet adhesiveness probably after incorporation by plasma lipoproteins.

Periosteally derived osteoblast-like cells differentiate into chondrocytes in suspension culture in agarose.

Pluripotent cells from the periosteal layer adjacent to cortical bone attain an osteoblast-like phenotype in culture when reaching confluence in monolayer. It is unknown whether such newly differentiated osteoblast-like cells preserve the chondrogenic potential characteristics for stem cells derived from the periosteum. Primary osteoprogenitor cells derived from bovine metacarpal periosteum were differentiated into alkaline phosphatase-positive osteoblast-like cells by an established monolayer culture protocol. After transfer into suspension culture in agarose gels, the cells differentiated into chondrocytes demonstrated by the production of collagen II, but not of collagen I, as well as alkaline phosphatase activity was abated. Contrarily, with continuation of monolayer culture, the cells maintained their osteoblast-like phenotype and secreted large amounts of collagen I and a minor quantity of collagen III and V. The alkaline phosphatase activity steadily increased during the entire culture period of 2 weeks. Thus, our culture techniques can serve as useful tools to study mechanisms of differentiation by modulating the phenotypic potential of osteogenic cells. The results presented here support the notion that the extracellular environment strongly influences the cell type and its metabolism.

Oxidized low-density lipoprotein inhibits the binding of monoclonal antibody to platelet glycoprotein IIB-IIIA.

Previous studies have shown that oxidized low-density lipoprotein (LDL) induces platelet activation more effectively than native LDL. To achieve a better understanding of the mechanism underlying the activation of human platelets by oxidized LDL, the present study relates the effect of oxidized LDL to changes of binding characteristics for glycoprotein (GP) IIb-IIIa. Washed human platelets were treated by monoclonal antibody against GP IIb-IIIa, and the ligand-receptor complexes were revealed by immunocytochemical techniques on the ultrastructural level. The localization of the antiglycoprotein IIb-IIIa was time-dependent. After binding to the platelet surface membrane and open canalicular system, the surface-membrane labeling decreased during longer incubation periods. Preincubation with oxidized LDL inhibited the binding of antiglycoprotein IIb-IIIa. Our findings suggest that GP IIb-IIIa acts as a receptor for oxidized LDL. The binding of oxidized LDL to the GP IIb-IIIa might be the first step in platelet activation by plasma lipoproteins.

Vitamin E (alpha-tocopherol) does not inhibit platelet stimulation by oxidized low density lipoprotein in vitro.

Platelet-rich plasma were treated with increasing concentrations of vitamin E (alpha-tocopherol). Washed platelets were exposed to oxidized low density lipoprotein (LDL) and examined by aggregometry and electron microscopy. The treatment of washed platelets by oxidized LDL induced morphological signs of activation like pseudopodia formation and an increase in light transmission. Alpha-tocopherol in a range of 0.001-1.0 mmol had no inhibiting influences on platelet activation by oxidized LDL. These results indicate that the free radical scavenger vitamin E cannot directly inhibit platelet activation by oxidized LDL. It may be supposed that platelet activation by oxidized LDL does not occur in a radical-dependent mechanism.

Ultrastructural aspects of cartilage formation, mineralization, and degeneration during primary antler growth in fallow deer (Dama dama).

Due to their rapid growth, regular replacement and easy accessibility, deer antlers are considered a useful model for the study of cartilage and bone differentiation and mineralization in mammals. The present study describes, for the first time, the cellular and extracellular matrix changes associated with cartilage formation, mineralization and degeneration in primary antlers on the ultrastructural level. Growing primary antlers of 3 to 4 cm length were obtained from six fallow bucks, aged about 10 months. It was shown that the chondroblasts were derived from progenitor cells of the antler perichondrium and differentiated into mature chondrocytes that subsequently underwent hypertrophic changes. Concomitant with cell hypertrophy, formation of a lacunar and a perilacunar extracellular matrix was observed, the latter containing numerous collagenous fibers. Mineralization of the extracellular matrix occurred via matrix vesicles and the formation of apatite crystals at distinct sites of the collagenous fibers. The hypertrophic chondrocytes of the mineralized cartilage then degenerated, a process that was also occasionally observed in more distally located cells surrounded by still unmineralized matrix. No morphological indications of a transdifferentiation of hypertrophic chondrocytes into bone forming cells, i.e., co-occurrence of a degenerating chondrocyte and a viable osteogenic cell in intact lacunae, were found. The cellular and extracellular matrix changes seen in primary antlers resemble those described for secondary antlers. Our results further indicate that the hypertrophic chondrocytes of primary antlers eventually undergo apoptosis, thereby providing further evidence that metaplastic conversion of cartilage into bone does not play a role in antler growth.

The mineralization of mantle dentine and of circumpulpal dentine in the rat: an ultrastructural and element-analytical study.

The purpose of this study was to compare the biomineralization of circumpulpal dentine with that of mantle dentine by ultrastructural and element-analytical techniques. Forty upper second molar germs of 10-day-old albino rats were cryofixed in liquid nitrogen-cooled propane and embedded in resin after freeze drying. Semithin dry sections were cut for analyzing the calcium and phosphorous concentration in initial mantle dentine, at the mineralization front of circumpulpal dentine, in the middle region of circumpulpal dentine and in mantle dentine peripheral to circumpulpal dentine. For the morphological evaluation of mineral deposits we compared ultrathin and unstained sections of cryofixed molars with chemically fixed molars. For both dentine types it was found that they develop via identical steps of mineral formation at collagen fibrils and non-collagenous matrix molecules. In circumpulpal dentine no globular mineral protrusions along the mineralization front (i.e. calcospherites) and no indications of interglobular dentine at the transition from circumpulpal dentine to mantle dentine were present. Two von Korff fibres were not only visible in mantle dentine but also in circumpulpal dentine. Matrix vesicles were present only during the formation of an initial coherent layer of mantle dentine and could not be observed during successive formation of mantle dentine and circumpulpal dentine. The element-analytical data did not demonstrate any difference in the mineral content between the two dentine types. Therefore, we conclude that mantle dentine and circumpulpal dentine in the rat molar possess a high degree of structural and chemical similarity and that only the extent of terminal branching of the odontoblast processes gives an approximate estimation of the thickness of mantle dentine.

Magnesium in newly formed dentin mineral of rat incisor.

Small amounts of magnesium are always detectable in addition to calcium and phosphorus in mineralized tissues such as dentin or bone. Magnesium has been considered to influence the mineralization process, especially crystal growth. The present study reports on the location and enrichment of magnesium in the newly mineralized dentin by using the high lateral resolution of energy dispersive X-ray microanalysis combined with scanning transmission electron microscopy. To this end, we have used the continuously growing rat incisor as a model for a collagenous mineralizing system. Dental tissue was dissected free and cryofixed in liquid nitrogen-cooled propane. The distribution of elements was measured in freeze-dried ultrathin cryosections. The magnesium distribution of the newly formed dentin area near the predentin area was found to be inhomogeneous. In certain small dentin areas, characteristical magnesium enrichments were observed. Further, high magnesium-to-phosphate molar ratios were found in these areas, and these were correlated with low calcium-to-phosphate molar ratios. Our results support the theory that magnesium is involved in the process of biological apatite crystal formation.

Tissue differentiation and correlated changes in enzymatic activities during primary antler development in fallow deer (Dama dama).

BACKGROUND: Deer antlers are useful models for studying bone growth and biomineralization in mammals. To achieve a better understanding of the mechanisms underlying the formation of primary cranial appendages in deer, the present study relates the histogenesis of primary antlers to changes in enzymatic (phosphatase) activities in the different tissue zones of this organ. METHODS: The growing tips of the primary antlers (4.3 to 5 cm in length) were removed from five fallow bucks, aged about 10 months. Part of the material was processed for light microscopy. The other part was cryofixed, and the different histologically defined regions were analyzed for the activities of alkaline phosphatase (ALP) and tartrate-resistant acid phosphatase (TRAP) as well as for the concentrations of inorganic and organic phosphate. RESULTS AND CONCLUSIONS: Histologically, the primary antler could in distoproximal direction be divided into eight different zones (dermis; perichondrium; zones of cartilage formation, hypertrophy, mineralization, and degeneration; primary spongiosa; secondary spongiosa). The histological results demonstrate that the elongation of the primary antler proceeded through a modified form of endochondral ossification, resembling that seen during formation of pedicles and secondary antlers. The concentrations of the extractable activities of ALP and TRAP progressively increased from the perichondrium to the zone of cartilage mineralization. Thus, highest activity of TRAP during primary antler formation occurred at an earlier stage of tissue differentiation than in somatic endochondral ossification, where the enzyme is a biochemical marker of osteoclastic activity during bone remodeling. The present results might reflect the presence of osteoclastic precursor cells in the zone of cartilage mineralization as an adaptation to the rapidity of antler growth. Our findings of the contents of extractable ALP, inorganic and organic phosphate in the different tissue zones of the developing primary antler are in good agreement with previous studies analyzing epiphyseal growth plates and point to the fact that ALP causes a rise in inorganic phosphate and the removal of inhibitors for mineralization, like pyrophosphate.

Biochemical and histological study of the ossification in the early developing pedicle of the fallow deer (Dama dama).

To date, no histochemical data exist concerning the process of ossification of developing pedicles in deer. Four different zones of the growing pedicle (subcutaneous tissue; fibrous layer of the periosteum; cambial layer of the periosteum; woven bone of the primary spongiosa) were analysed in direct correlation to their histological appearance. The level of extractable specific alkaline phosphatase in the preosseous zones of the pedicle was 4-fold higher than levels in the epiphyseal growth plate previously reported. These results reflect that rapid bone formation takes place in the growing pedicle. Highest buffer-extractable alkaline phosphatase activity was found in the cambial layer directly in front of the mineralization area of the pedicle-bone, connected with maximal values for organically bound phosphate and inorganic phosphate. Moreover, the values for buffer-extractable alkaline phosphatase, organically bound phosphate and inorganic phosphate decreased with increasing mineralization in the zone of the primary spongiosa. The present histological and biochemical findings on the process of ossification in the pedicle show similarities to typical endochondral ossification. The process of pedicle growth may serve as a new and important system for chondrogenic and osteogenic studies, including a better understanding of antler development.

Light microscopic observations on the ossification process in the early developing pedicle of fallow deer (Dama dama).

The ossification process of the early developing pedicle was studied in five male fallow deer fawns, aged about seven months. The incipient pedicle was covered by a periosteum, the cambial layer of which was significantly thicker at the apex of the outgrowth than in the more peripheral areas of the pedicle. As was demonstrated histologically, in the central part of the pedicle elongation occurred by a process corresponding to endochondral ossification, whereas in the more peripheral areas the pedicle became enlarged by typical intramembranous ossification. Thus, cartilage formation must be regarded as a normal feature in pedicle growth of fallow deer. The assumption that the transition from pedicle to first antler growth in cervids is reflected by a switch from intramembranous ossification to chondrogenesis at the apex of the growing primary cranial appendage, based mainly on observations in roe deer, does, therefore, not hold for fallow deer. Furthermore, histogenesis of the central part of the fallow deer pedicle closely resembles the developmental events leading to formation of subsequent antlers.