Pentose-shunt oxidation in the periosteal cells in healing fractures

Summary The activity of pentose-shunt dehydrogenases is very low in periosteal cells of normal rat metatarsals, but increases one day post-fracture and rises linearly over the next two days. By four days postfracture, the distribution of this activity along the bone shows two centres of high activity: the first in the region of proliferation to form callus and the second at the site where new bone is first seen, one day later. The high rate of generation of NADPH would be expected to reduce glutathione; reduced glutathione has been shown to inhibit alkaline phosphatase activity in these cells.     

Low intensity pulsed ultrasound stimulates osteogenic activity of human periosteal cells

The effect of low intensity pulsed ultrasound on human periosteal cells was investigated. Normal human periosteum was obtained to culture the periosteal cells. After characterization, cultures of periosteal cells at Days 2 and 4 were treated with ultrasound for 5, 10, and 20 minutes respectively. Assessments were done to assess total number of viable cells, cell proliferation, alkaline phosphatase activity, osteocalcin secretion, vascular endothelial growth factor expression, and calcium nodule formation. With the cells not treated with ultrasound as the control, the results showed that ultrasound did not affect the total number of viable cells. It stimulated cell proliferation at the early phase of cell culture. The activity of alkaline phosphatase was increased significantly in the culture at Day 4. A similar effect was seen with osteocalcin secretion and the responses were dose-dependent. The vascular endothelial growth factor secretion increased in Day 2 and Day 4 cultures with the dose-dependent effect. Formation of calcium nodules was significantly higher with ultrasound treatment. We think that low intensity pulsed ultrasound stimulated periosteal cell proliferation and differentiation toward osteogenic lineage. The dose-dependent effect on osteogenic activities may modify the existing treatment regimen. Ultrasound treatment should be started from the beginning of fracture healing.     

Alkaline phosphatase production by periosteal cells at various oxygen tensions in vitro

A mammalian periosteal cell culture system was developed to investigate the metabolic response of fresh calf bone periosteal cells to various oxygen tensions in vitro. Two predominant cell phenotypes were seen in the culture system. A rapidly proliferating mat of alkaline phosphatase-negative cells supported the growth of overlying clusters of alkaline phosphatase-positive cells. The appearance and subsequent population growth of the alkaline phosphatase-positive cells correlated directly with increases in enzyme activity on biochemical assay. Alkaline phosphatase production was optimal at lower oxygen tensions (5%, 9%), which approximated capillary pO2. In addition, the preconfluence oxygen environment was more critical to the final expression of the enzyme activity than the postconfluence environment. The mechanism of the environmental regulation of alkaline phosphatase gene expression at various oxygen tensions is not known. Periosteal cells were highly sensitive to oxygen tension and expressed alkaline phosphatase enzyme activity at oxygen levels approximating capillary rather than atmospheric pO2.     

Osteogenic capacity of cultured human periosteal cells

We developed a culture system of cells isolated from juvenile human periosteum. The culture consisted of epithelial-like and fibroblast-like cells. Both types of cells had intense alkaline phosphatase activity maintained in subculture. When these cells, loaded into diffusion chambers, were implanted subcutaneously in rats, cartilage tissue was mainly formed and bone was seen scantily. 1 alpha-OH-D3 and 1,25-(OH)2-D3 increased the alkaline phosphatase activity and proteoglycan synthesis in the periosteal cells. Calcitonin also stimulated the proteoglycan synthesis, but parathyroid hormone had no effect.     

Evidence for bone formation on the external "periosteal" surface of the femoral neck: a comparison of intracapsular hip fracture cases and controls

Age-related expansion of the external surface of the femoral neck in order to offset generalized bone loss is potentially an important mechanism whereby hip strength and hence resistance to hip fracture is maintained. However, it has been widely assumed that bone formation is precluded from this external interface due to the presence of a synovial membrane associated with the hip joint. In this study we have demonstrated histologically that bone formation does indeed occur on the outer "periosteal" surface of the proximal femoral neck. It was therefore hypothesized that an impairment or reduction in periosteal bone formation might be seen in cases of femoral neck fracture compared with age-matched controls. Qualitative analysis of whole femoral neck samples from female subjects and age- and sex-matched post-mortem controls demonstrated that these groups expressed similar distributions of the bone formation marker, alkaline phosphatase (AP), at the periosteal surface [whole biopsy mean % periosteal AP-positive surface: control=16.0 (range=0.5-43.0), fracture=13.4 (range=1.0-34.6), p=0.44]. In conclusion, despite a wide intersubject variation, bone formation at the femoral neck periosteum is a feature of elderly women even if they have had a hip fracture.     

Initial effects of partially purified bone morphogenetic protein on the expression of glycosaminoglycan, collagen, and alkaline phosphatase in nonunion cell cultures.

Bone morphogenetic protein (BMP) stimulates mesenchymal cells to differentiate, resulting in de novo endochondral ossification in vivo. The response of fibrocartilage and periosteal cells from human and canine nonunion tissues to partially purified BMP was examined in culture. Cells derived from neonatal rat muscle explants were used for comparison. Alkaline phosphatase activity and expression of alkaline phosphatase and Types I and II collagen mRNAs were compared to that of rat chondrocytes. Synthesis of Type II collagen by the muscle cells was verified by enzyme-linked immunosorbent assay (ELISA). Addition of BMP to the muscle cell and nonunion cell cultures resulted in a dose-dependent decrease in cell number. There was a decrease in matrix vesicle and plasma membrane alkaline phosphatase activity concomitant with an increase in mRNA levels for alkaline phosphatase and collagen genes. Synthesis of immunoreactive Type II collagen increased. These data indicate that neonatal rat muscle cells and nonunion cells may respond in a similar fashion to BMP. Bone morphogenetic protein stimulated hyaluronic acid synthesis at three days, but chondroitin sulfate synthesis did not increase until ten days exposure to BMP. These data, together with those summarized above, suggest that more than three days may be required for complete expression of the chondrocyte phenotype typical of endochondral ossification.     

Effect of cortisol on periosteal and nonperiosteal collagen and DNA synthesis in cultured rat calvariae

Summary The effects of cortisol on bone formation are complex and may be modulated by the presence of periosteal cells or by factors released by the periosteal tissue. To test these possibilities, cortisol was examined for its effects on the incorporation of³H-proline into collagenase-digestible protein (CDP) and noncollagen protein (NCP), on DNA synthesis and on alkaline phosphatase activity in intact and in the periosteum and nonperiosteal bone of dissected calvariae from 21-day-old fetal rats. After 24 h of treatment, cortisol increased the incorporation of³H-proline into CDP in intact bones and in the nonperiosteal bone of calvariae dissected after the culture. Cortisol inhibited the incorporation of³H-thymidine into calvarial DNA but it caused a small increase in nonperiosteal DNA content. Cortisol did not affect the incorporation of³H-proline into CDP in calvariae dissected prior to the culture if the periosteum and nonperiosteal central bone were incubated separately; the stimulatory effect was observed only if the two tissues were cultured in the same vial and were in contact. In contrast, cortisol stimulated alkaline phosphatase activity in the central nonperiosteal bone of calvariae dissected before or after the culture. After 72–96 h of treatment, cortisol inhibited the labeling of CDP, NCP, and DNA and the DNA content in intact bones and in both periosteal and nonperiosteal central bone of calvariae dissected after the culture. In contrast, when the periosteum was removed before the incubation, these inhibitory effects were observed in the periosteum and not in the nonperiosteal bone. Cortisol inhibited alkaline phosphatase activity in intact bones treated for 96 h, but removal of the periosteum resulted in a stimulatory effect in the nonperiosteal central bone. These studies indicate that 24 h treatment with cortisol stimulates collagen synthesis in nonperiosteal bone, an effect that requires the presence of periosteal tissue. Exposure to cortisol for 72–96 h inhibits collagen, noncollagen protein, and DNA synthesis, an effect that is secondary to an inhibition of periosteal cell replication. Cortisol does not cause a direct inhibition of osteoblastic function.     

Distribution of alkaline phosphatase activity in experimentally produced callus in rats

Experimentally produced fractures in long bones studied by light and electron microscopic histochemistry were found to heal by a process of enchondral calcification. There was intense proliferation in the cells of the cambium layer of the periosteum, with differentiation to chondroblasts and osteoblasts, suggesting that this layer was the primary tissue responsible for development of the callus. Cytoplasmic processes of the hypertrophic chondrocytes appeared to bud and produce matrix vesicles. Alkaline phosphatase activity was detected along the plasma membrane of the hypertrophic chondrocytes and around the matrix vesicles, before any signs of mineral deposition. Calcification took place by deposition of hydroxyapatite crystals in and around these matrix vesicles which frequently showed alkaline phosphatase activity. It is suggested that there is a close functional association between alkaline phosphatase activity and calcification in the process of fracture healing, which is another type of enchondral calcification mediated by matrix vesicles.     

Effects on fracture healing of an antagonist of the vitamin K cycle

Summary The anticoagulant, dicumarol, inhibits the vitamin K cycle by blocking the conversion of the vitamin K epoxide. The effects of dicumarol on ossification have been tested by feeding it to rats in which a closed fracture of the metatarsals had been induced; the effects were studied up to 12 days postfracture. At 12 days, treatment with dicumarol caused a highly significant decrease in the amount of bone produced, without affecting the total size of the callus. Quantitative cytochemistry of unfixed, undemineralized sections showed that dicumarol also markedly affected the periosteal activities of glucose 6-phosphate dehydrogenase and of alkaline phosphatase in the first 2 mm from the fracture measured at 3 and 5 days postfracture when normally, new bone is first formed. In contrast, dicumarol had little effect on these activities in the fully formed callus.     

成骨因子BMP7在骨膜细胞体外培养中的作用

目的 探讨成骨因子骨形态发生蛋白(BMP7)在骨膜细胞体外培养中的诱导分化作用.方法取材于成人胫骨骨膜,常规细胞培养法行骨膜细胞体外培养,分为实验组和对照组,分别加入BMP7加成骨细胞培养辅助剂和单纯成骨细胞培养辅助剂,相差显微镜观察骨膜细胞形态特征.每组分别在第5、10、15、20天设3个样本,采用ALP试剂盒法及钙结节Von Kossa染色法分别检测成骨细胞特异性标志物ALP和钙结节的表达情况.结果 骨膜细胞在体外生长良好,实验组和对照组形成的成骨细胞增殖良好,形态一致.细胞早期呈梭形,饱满透明,立体感强;分裂期呈立方形或短柱状;后期由长梭形逐渐变成宽梭和不规则形.由BMP7诱导的骨膜细胞的成骨标志物ALP和钙结节染色阳性率明显高于对照组,有统计学意义(P＜0.01).结论 骨膜细胞具有良好的成骨和再生能力,BMP7在体外培养中具有明显增强骨膜细胞分化为成骨细胞的作用.     

A monoclonal antibody against the surface of osteoblasts recognizes alkaline phosphatase isoenzymes in bone, liver, kidney, and intestine

Monoclonal antibodies against the surface of embryonic osteogenic cells have been used to characterize the osteoblastic lineage. One antibody, SB-1, reacts in frozen sections with a family of cells in bone, liver, kidney, and intestine which are identically stained by the histochemical substrate for alkaline phosphatase. In this report, biochemical and immunochemical evidence is presented to indicate that SB-1 is directed against an epitope on alkaline phosphatase which is shared by isoenzymes in a variety of chick tissues. In a solid-phase assay system, high dilutions (1:10(5] of ascites fluid were found to give significant binding of SB-1 to alkaline phosphatase extracted from chick limb or intestine. Partial purification of intestinal alkaline phosphatase on a Sepharose CL-6B column results in the co-elution of alkaline phosphatase enzyme activity and antibody-binding material; this indicates that SB-1 recognizes intestinal alkaline phosphatase rather than an impurity in the crude preparation. Furthermore, Western immunoblots of chick calvarial bone extract electrophoresed on a 5-20% SDS-polyacrylamide gel show that SB-1 reacts with a single 155 kD band which also is stained by the alkaline phosphatase histochemical substrate. In a similar set of experiments, SB-1 reacts with an intestinal alkaline phosphatase isoenzyme whose molecular weight is approximately 185 kD. From these studies, we conclude that SB-1 is specifically reactive with alkaline phosphatase isoenzymes present in bone, liver, kidney, cartilage, and intestine. The reactive epitope is stable to SDS denaturation, not associated with the active site of the enzyme, and dependent on disulfide bonds which impart secondary structure to the protein.     

The cartilaginous fracture callus in rats

The right tibia was broken manually in 56 rats weighing 100 g; the fracture was stabilized with an intramedullary steel wire. Groups of rats were killed after 3-30 days. The fracture with its surrounding musculature was dissected out and immediately frozen to -70 degrees C. Cryostat sections of the fracture region were stained with hematoxylin/eosin, toluidine blue, and immunoenzymatically with collagen II antibodies. Another series of 30 fractured rats were killed after 1-15 days, and the fractures were examined histologically after decalcification with EDTA. Two types of callus were observed. The periosteal-endosteal callus started as proliferation of pre-osteoblasts without inflammatory cells on Day 1 and developed bone trabeculae from Day 3 until Days 8-10, but not thereafter. The cartilaginous callus was formed by condensation of fibroblast-like mesenchymal cells mixed with inflammatory cells outside the periosteal callus and started on Day 5 at the fracture fragments denuded of periosteum. The cells differentiating to cartilage seemed to migrate from the surrounding musculature and its newly formed vessels. The enchondral bone formation started close to the periosteal callus from which vessels were piercing into the then hypertrophic and mineralized chondrocytes on Day 11. We conclude that the bridging callus is formed by fundamentals of periosteal callus derived from predetermined cells and the bridge of enchondrally formed bone trabeculae by cells migrating from outside.     

The cartilaginous fracture callus in rats

The right tibia was broken manually in 56 rats weighing 100 g; the fracture was stabilized with an intramedullary steel wire. Groups of rats were killed after 3-30 days. The fracture with its surrounding musculature was dissected out and immediately frozen to -70° C. Cryostat sections of the fracture region were stained with hematoxylin/eosin, toluidine blue, and immunoenzymatically with collagen II antibodies. Another series of 30 fractured rats were killed after 1-15 days, and the fractures were examined histologically after decalcification with EDTA.

Two types of callus were observed. The periosteal-endosteal callus started as proliferation of preosteoblasts without inflammatory cells on Day 1 and developed bone trabeculae from Day 3 until Days 8-10, but not thereafter. The cartilaginous callus was formed by condensation of fibroblastlike mesenchymal cells mixed with inflammatory cells outside the periosteal callus and started on Day 5 at the fracture fragments denuded of periosteum. The cells differentiating to cartilage seemed to migrate from the surrounding musculature and its newly formed vessels. The enchondral bone formation started close to the periosteal callus from which vessels were piercing into the then hypertrophic and mineralized chondrocytes on Day 11.

We conclude that the bridging callus is formed by fundaments of periosteal callus derived from predetermined cells and the bridge of enchondrally formed bone trabeculae by cells migrating from outside.     

Neutral protein-degrading enzymes in experimental fracture callus: a preliminary report

The process of endochondral fracture healing is biochemically similar to growth plate calcification. Recent studies have identified potentially important roles for proteoglycan-degrading enzymes in the growth plate. The purpose of the study described herein was to identify, in healing fractures, neutral enzyme activities capable of degrading proteoglycans and other matrix proteins. Two sets of 60 male Sprague-Dawley rats underwent the production of closed femoral fractures. Calluses were retrieved at timed intervals, and cell and matrix vesicle fractions were prepared for electron microscopy, neutral peptidase, and alkaline phosphatase assays. In another group of 10 animals, fractions were prepared from 14-day calluses and examined for proteoglycanase activity. In the cell fractions, alkaline phosphatase, alanyl-beta-naphthylamidase, aminopeptidase, and endopeptidase activities showed somewhat parallel distributions peaking at approximately 14-17 days. In the matrix vesicle fractions, similar relative distributions were observed for alkaline phosphatase and endopeptidase. However, here the peak activities occurred up to 3 days later than they did in the cell fractions. Significant proteoglycanase activity was confirmed in both cell and matrix vesicle fractions. These findings are consistent with the hypotheses that (a) neutral peptidases, by virtue of their temporal expression in parallel with alkaline phosphatase, may be involved in preparing fracture callus matrix for calcification; and (b) matrix vesicles may convey certain of these enzymes to sites of both matrix degradation and calcification, since the same activities found in cells are found in matrix vesicles a few days later. The possibility that some of these enzymes are involved in growth factor activation remains to be investigated.     

Circulating cells with osteogenic potential are physiologically mobilized into the fracture healing site in the parabiotic mice model

Based on the accumulating evidence of osteogenic cells present in the systemic circulation, we hypothesized that circulating osteogenic connective tissue progenitors (CTPs) home to fracture site and contribute to skeletal repair. Parabiotic animals were formed by surgically conjoining transgenic mice constitutively expressing green fluorescent protein (GFP) in no erythroid tissue and syngeneic wild‐type mice. After 3 weeks parabionts, equilibrium in blood chimerism between partners was established. A transverse fibular fracture was made in the contralateral hind limb of the conjoined wild‐type partner. The contribution of circulating cells to the fracture callus was assessed based on analysis of GFP+ cells and co‐localization of alkaline phosphatase (AP) staining nonfracture and at 1, 2, 3, and 4 weeks after fracture. Histomorphometric analysis at the fracture site showed significant increase of GFP+ cells after 2 (5.4%) and 3 (5.6%) weeks compared to nonfractured controls (1.7%). Of the GFP+ cells, percentage of the cells expressing AP activity at 1 (37.4%) and 2 (85.3%) weeks postfracture time was statistically higher than that in nonfractured controls (10.8%). The rate of mobilization of circulating osteogenic CTPs to fracture callus was also examined using 1 week parabionts at week 0–1 and week 1–2 postfracture. There was significant increase of GFP+/AP+ cells from week 0–1 (0.1%) and week 1–2 (1.8%). These data indicate that circulating osteogenic CTPs are mobilized to fracture site and contribute to osteogenesis in the early stage of fracture healing. © 2007 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 26:165–175, 2008     

Osteosarcoma cells in tissue culture: II. Characterization and localization of alkaline phosphatase activity

Although elevated alkaline phosphatase levels in osteosarcoma have been shown to be related to prognosis, the functional significance is unclear. Human osteosarcoma cells in tissue culture retain detectable amounts of alkaline phosphatase activity. In this study the specific activity of that enzyme was compared in 13 osteosarcoma tissue culture lines and 13 normal skin fibroblast lines derived from the same patients. Osteosarcoma cells had significantly higher baseline alkaline phosphatase levels and could be stimulated with hydrocortisone to produce more enzymatic activity than the fibroblast lines. Activity was localized ultracytochemically to the cell membrane and to many small intracellular vesicles in stimulated osteosarcoma cells. These observations aid in the differentiation of osteosarcoma and fibroblast lines in tissue culture and suggest an association between elevated alkaline phosphatase levels and metabolic abnormalities in patients with osteosarcoma.     

Proliferation and macromolecular synthesis by rat calvarial bone cells grown in various oxygen tensions

Perinatal rat calvarial bone cells were isolated by sequential collagenase digestion and grown in oxygen tensions ranging from 1 to 60% O2. Cell proliferation as determined by automated cell counting and DNA content was greatest in the lower oxygen tensions (less than or equal to 9% O2), whereas alkaline phosphatase activity and [35S]sulfate and [14C]proline incorporation were greatest in the higher oxygen tensions (greater than or equal to 13% O2). It is concluded that lower oxygen concentrations favor bone cell proliferation, whereas higher oxygen concentrations favor macromolecular synthesis. These findings, when related to the known pO2 of the fracture callus, suggest the following sequence of events: first, at the time of fracture an ingrowth of osteoprogenitor cells, capillary buds, and primitive mesenchymal cells occurs in the fracture site, a region of low pO2; second, a great increase in cellular proliferation accompanied by an initiation of macromolecular synthesis follows; finally, as the pO2 levels begin to increase, cellular proliferation decelerates, accompanied by an increase in macromolecular synthesis.     

Mechanobiology of initial pseudarthrosis formation with oblique fractures.

Mechanical stresses play an important role in regulating tissue differentiation in a variety of contexts during skeletal development and regeneration. It has been shown that some intermittent loading at a fracture site can accelerate secondary fracture healing. However, it has not been shown how the stress and strain histories resulting from mechanical loading of a fracture might, in some cases, inhibit normal fracture healing and induce pseudarthrosis formation. In this study, finite element analysis is used to calculate hydrostatic stress and maximum principal tensile strain patterns in regenerating tissue around the site of an oblique fracture. Using a mechanobiologic view on tissue differentiation, we compared calculated stress and strain patterns within the fracture callus to the histomorphology of a typical oblique pseudarthrosis. Tissue differentiation predictions were consistent with the characteristic histomorphology of oblique pseudarthrosis: in the interfragmentary gap. tensile strains led to "cleavage" of the callus; at the ends of both fracture fragments, hydrostatic pressure and tensile strain caused fibrocartilage formation, and, at discrete locations of the periosteum at the oblique fracture ends, mild hydrostatic tension caused bone formation. We also found that discrete regions of high hydrostatic pressure correlated with locations of periosteal bone resorption. When previous findings with distraction osteogenesis are considered with these observations, it appears that low levels of hydrostatic pressure may be conducive to periosteal cartilage formation but high hydrostatic pressure may induce periosteal bone resorption during bone healing. We concluded that tissue differentiation in pseudarthrosis formation is consistent with concepts previously presented for understanding fracture healing, distraction osteogenesis, and joint formation.     

Role of fracture hematoma and periosteum during fracture healing in rats: interaction of fracture hematoma and the periosteum in the initial step of the healing process

To study the mechanisms of fracture healing, we investigated the interaction between fracture hematoma and periosteum during the early phase of fracture healing in rats. Experimentally induced fractures of the tibia in untreated rats were compared histologically with such fractures in rats in which either the bone marrow or the periosteum had been removed. The extent of periosteal cell proliferation and chondrogenesis in the fracture hematoma was evaluated on experimental days 3, 6, 10, and 14. On day 3, periosteal cell proliferation at the tibial fracture site was decreased in the bone marrow-removed rats compared with the proliferation in untreated rats. Little chondrogenesis in the fracture hematoma was seen through day 6 in the periosteum-removed rats. These results suggest that the periosteum is important for mediating the primary steps of chondrogenesis and enchondral ossification in the fracture hematoma and that the fracture hematoma may be essential for periosteal cell proliferation during fracture healing. Received for publication on April 5, 1999; accepted on July 21, 1999