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Biomedical subjects

I Binderman

Publications and source records attributed to I Binderman.

At least 19 recordsLinked to original sources

Effect of low intensity laser irradiation on surgically created bony defects in rats.

Low intensity lasers have been used by clinicians to improve healing and reduce pain in humans. Lasing also results in new bone formation around hydroxyapatite implants and a significant increase in the total bone area. However, the exact mechanism of cell biostimulation by laser is still unclear. This study biochemically assessed the effects of low intensity laser (Gallium-Arsenide) using 4 and 22.4 mW cm(-2) power density on the bone healing process after surgically creating bony cavities in rat mandibles. Rats (n = 24) were divided into two groups each treated with specific energy, 4 or 22.4 mW cm(-2), for 3 min each day post-surgery. Surgical cavities were created on both sides of the mandible: the left served as an untreated control, the right was treated with laser. All rats were sacrificed after 1, 2 and 4 weeks of treatment. In the newly formed callus, accumulation of radiocalcium and alkaline phosphatase activity was measured to indicate osteogenic activity. One-way anova with repeated measures showed that the low intensity laser using 4 mW cm(-2) power density significantly increased radiocalcium accumulation from 2 weeks post-surgery, whereas 22.4 mW cm(-2) had no effect. No changes were noted in the activity of alkaline phosphatase with the laser treatment. These results suggest that laser therapy of low power density is effective on the bone healing process in artificially created osseous cavities by affecting calcium transport during new bone formation.

Alkaline Phosphatase↗

Combined local application of tetracycline and bisphosphonate reduces alveolar bone resorption in rats.

BACKGROUND: Recent animal studies have shown that a combination of chemically-modified tetracyclines together with bisphosphonates, when delivered systemically, are synergistically effective in suppressing periodontal bone loss. In the present study, we explored the combined efficacy of local delivery of alendronate and tetracyclines in reducing alveolar bone loss. METHODS: Eighty-six (86) male Wistar rats were used in these experiments. The flap was elevated using a special periosteal elevator, on both sides of the mandible, as described previously. A gelfoam pellet containing the drugs was applied between the alveolar bone and the mucoperiosteal flap, according to the experimental protocol. The rats were divided into 5 treatment groups: 1) alendronate; 2) doxycycline hyclate 10% (DOXY); 3) tetracycline hydrochloride 1% (TET); 4) alendronate + DOXY; and 5) alendronate + TET. In the operated control sites (C), saline was applied. The rats were sacrificed 21 days following the flap procedure. Sections of the mandibles (1.5 mm), in a buccal-lingual direction, underwent microradiography and were analyzed for bone loss. RESULTS: DOXY alone was most effective in reducing bone loss. Alendronate was also effective in reducing bone loss as shown in previous reports. TET did not reduce bone loss significantly when used alone. In combination with alendronate TET was synergistically effective. The combined local treatment of alendronate + DOXY showed no additive effect. CONCLUSIONS: In the present study, we found that tetracyclines can be most effective in reducing alveolar bone loss when applied locally. The combined local treatment of alendronate and tetracycline may have a synergistic effect.

Administration, Topical↗

BMP-6 accelerates both chondrogenesis and mineral maturation in differentiating chick limb-bud mesenchymal cell cultures.

Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate in vitro to form a cartilaginous structure analogous to the epiphyseal growth plate. When inorganic phosphate, Pi, is included in the medium such that the total Pi concentration is 4 mM, apatite mineral precipitates around the "hypertrophic" chondrocytes. These hypertrophic chondrocytes are characterized by their increased expression of type X collagen, alkaline phosphatase activity, and apoptosis, as well as by the ability of their extracellular matrices to support mineral deposition. Under standard mineralizing conditions (0.8 x 10(6)cells/micromass; 4 mM Pi, 1.3 mM Ca(2+), 10% FCS, and antibiotics) mineralization does not commence until day 14-16. Based on the ability of bone morphogenic protein 6 (BMP-6) to stimulate chondrocyte maturation in other systems, 100 ng/ml BMP-6 was added to chick limb-bud mesenchymal cell cultures 2 and 5 days after plating, and the effects of this addition on mineral accretion and the characteristics of the mineral and matrix determined. Addition of BMP-6 accelerated the differentiation of the mesenchymal cells to hypertrophic chondrocytes. In the presence of BMP-6 added on both days 2 and 5, mineralization (assessed on basis of (45)Ca uptake) commenced by day 12. Fourier transform infrared imaging (FTIRI) was used to monitor the mineral content and mineral crystallinity as a function of time from day 9 to 21 in cultures with and without exogenous BMP-6. While BMP-6 accelerated the rate of mineral accretion, and the crystals that were formed in the BMP-6 cultures were initially more mature, by day 21 the crystal size distribution in experimental and control cultures were not significantly different. This study, the first to report the detailed application of FTIRI to cell cultures, indicates the importance of the extracellular matrix in the control of crystal maturation.

Alkaline Phosphatase↗

Alveolar bone resorption following coronal versus apical approach in a mucoperiosteal flap surgery procedure in the rat mandible.

BACKGROUND: In several publications, we have reported that a distinct resorptive phase of alveolar bone developed during 3 weeks following elevation of a mucoperiosteal flap in rats by a coronal surgical approach. In the present study, we compared the alveolar bone loss after mucoperiosteal flap surgery by a coronal approach (COR) to a surgical approach incising the mucosa close to the tooth apices and separating the mucoperiosteum toward the tooth crown (apical approach, AP). METHODS: Eighteen Wistar rats were divided into 2 experimental groups; each group consisted of 9 rats. In the first group, COR was performed on both buccal and lingual aspects on the right side of the rat mandible, while the left side served as a sham-operated control. In the second experimental group, AP was performed. RESULTS: In the AP group, the outer aspect (buccal or lingual) of the alveolar bone was slightly resorbed usually at the level where the mucosa was incised. In many sections, extensive modeling of new trabecular bone was seen, coronal to the surgical incision. On the contrary, the coronal surgical approach revealed an extensive resorptive phase, mainly of the periodontal aspect of the alveolar bone. CONCLUSIONS: The results of this study show that periodontal surgery by a coronal surgical approach stimulates a burst of remodeling of the alveolar bone in a very specific pattern.

Alveolar Bone Loss↗

Type I collagen influences cartilage calcification: an immunoblocking study in differentiating chick limb-bud mesenchymal cell cultures.

Chick limb-bud mesenchymal cells, plated in high-density micro-mass culture, differentiate and form a matrix resembling chick epiphyseal cartilage. In the presence of 4 mM inorganic phosphate or 2.5 mM beta-glycerophosphate mineral deposits upon this matrix forming a mineralized tissue that, based on electron microscopy, x-ray diffraction and Fourier Transform Infrared microspectoscopy, is like that of chick calcified cartilage. In this culture system the initial mineral deposits are found on the periphery of the chondrocyte nodules. During differentiation of the cells in the high-density micro-mass cultures there is a switch from expression of type I collagen to type II, and then to type X collagen. However, type I collagen persists in the matrix. Because there is some debate about whether type I collagen influences cartilage calcification, an immunoblocking technique was used to determine the importance of type I collagen on the mineralization process in this system. Studies using nonspecific goat anti-chick IgG demonstrated that 1-100 ng/ml antibody added with the media after the cartilage nodules had developed (day 7) had no effect on the accumulation of mineral in the cultures. Nonspecific antibody added before day 7 blocked development of the cultures. Parallel solution based cell-free studies showed that IgG did not have a strong affinity for apatite crystals, and had no significant effect on apatite crystal growth. Type I collagen antibodies (1-200 ng/ml) added to cultures one time on day 9 (before mineralization started), or on day 11 (at the start of mineralization), slightly inhibited the accumulation of mineral. There was a statistically significant decrease in mineral accretion with 100 or 200 ng/ml collagen antibody addition continuously after these times. Fab' fragments of nonspecific and type I collagen antibodies had effects parallel to those of the intact antibodies, indicating that the decreased mineralization was not attributable to the presence of the larger, bulkier antibodies. The altered accumulation of mineral was not associated with cell death in the presence of antibody (demonstrated by fluorescent labeling of DNA) or with increased apoptosis (TUNEL-stain). In the immunoblocked cultures, EM analysis demonstrated that mineral continued to deposit on collagen fibrils, but there appeared to be fewer deposits. The data demonstrate that type I collagen is important for the mineralization of these cultures.

Animals↗

The effect of topical delivery of novel bisacylphosphonates in reducing alveolar bone loss in the rat model.

BACKGROUND: Periodontal surgery stimulates osteoclast activity, leading to varying amounts of alveolar crest loss. We have established that topical application of 20 mg/ml of alendronate placed at the surgical mucoperiosteal site produced a striking reduction of alveolar bone loss in the rat model. The aim of this investigation was to examine the antiresorptive efficacy of 3 novel bisacylphosphonates topically delivered at the surgical site, in comparison to alendronate and etidronate which are in clinical use. METHODS: Mucoperiosteal flap (MF) surgery was performed on the buccal and lingual aspects next to molars on both sides of the rat mandible. A gelatin sponge soaked in the bisphosphonate solution prepared by dissolving 20 mg of the bisphosphonate (alendronate, etidronate, VS-5, VS-6, ISA-13, SuBP) in 1 ml of saline was applied to exposed bone on the right side of the mandible (experimental, MF + BPs ) and the left side was treated with saline only (control, MF + S). Sections were evaluated for bone loss using microradiography pattern and amount. RESULTS: The 3 novel bisacylphosphonates, VS-5 VS-6, and ISA-13 were more effective than etidronate, and less effective than alendronate. The most effective among this group was ISA-13 followed by VS-5 and VS-6. CONCLUSION: We conclude that ISA-13-like alendronate is effective in reducing alveolar bone loss when delivered at surgical sites. Since ISA-13 is well absorbed through mucose tissues, we suggest that ISA-13 efficacy on reducing bone loss should be tested by its application on the mucosal tissue.

Administration, Topical↗

Effectiveness of local delivery of alendronate in reducing alveolar bone loss following periodontal surgery in rats.

BACKGROUND: Mucoperiosteal flaps are used to access bone and root surfaces for debridement, pocket elimination, management of periodontal defects, and in regenerative procedures, as well as in implant surgery. Many reports show that periodontal surgery stimulates osteoclast activity with varying amounts of alveolar bone loss. Alendronate given intravenously significantly reduced alveolar bone loss in mucoperiosteal flap procedures. In the present study, we explored the effectiveness of different concentrations of alendronate, delivered at the surgical site at the time of surgery, in distant delivery in reducing alveolar bone loss. METHODS: Following elevation of a mucoperiosteal flap next to molars of the rat mandible, a gelatin sponge soaked with different concentrations of alendronate (0, 1, 5, 20, or 40 mg/ml; experiment A) was applied to exposed bone on the experimental side. In the second group (experiment B), alendronate (0, 50, 200, or 400 microg) was topically delivered in the cheek submucosa on the left side (distant to the surgical site) in a small cut into which the gelatin sponge soaked with the drug was placed. RESULTS: Topical application of 200 microg and 400 microg doses of alendronate at the time of surgery was significantly effective (P <0.001) in reducing bone loss. Generally, the percentage of sections with mild bone loss (V1, V2) increased with an increase in the dose of alendronate, while the percentage of sections with severe bone loss (H1, H2) decreased with an increase in alendronate dose. Topical application of 400 microg of alendronate had a systemic effect. CONCLUSIONS: This study implies that topical delivery of alendronate at the time of surgery reduces bone loss in periodontal procedures involving mucoperiosteal flap surgery. The most effective dose is 200 microg for topical delivery at the surgical site and 400 microg for distant sites.

Administration, Buccal↗

Disposition of alendronate following local delivery in a rat jaw.

BACKGROUND: Recently, we have shown that local delivery of alendronate reduced significantly bone resorption activated by surgical separation of periosteum from bone. These results advocate the use of local application of alendronate in bone surgeries to prevent regional bone resorption at the surgery site. Here we investigated the efficacy of absorbtion of alendronate by the bone from a gelatin sponge soaked with radiolabeled alendronate applied topically at the surgical site. METHODS: Following elevation of the mucoperiosteal flap next to premolars and molars of the rat mandible, a gelatin sponge soaked with 10 microl of radiolabled alendronate (1 microCi/mg) was applied to exposed bone on one side. The local absorbtion of alendronate and its disposition in the contralateral side of the mandible as well as in the tibia bone were analyzed. RESULTS: The results show that 10% of total alendronate content of the gelatin sponge was absorbed in the bone locally (in the surgical site), while 0.2% was disposed in the tibia. Of interest is the fact that the surgical wound in the contralateral side increased the disposition of alendronate up to 2%. This finding is most likely the result of extravasation and diffusion of alendronate due to surgical wounding. CONCLUSION: This study strongly supports our notion that local delivery of alendronate and its affinity to bone may become a very important treatment modality to prevent resorption of bone during dental and orthopedic procedures.

Absorption↗

Effects of proteoglycan modification on mineral formation in a differentiating chick limb-bud mesenchymal cell culture system.

In the presence of 4 mM inorganic phosphate, differentiating chick limb-bud mesenchymal cells plated in micromass cultures form a mineralized matrix resembling that of chick calcified cartilage. To test the hypothesis that cartilage proteoglycans are inhibitors of cell mediated mineralization, the synthesis, content, and turnover of proteoglycans were altered in this system, and the extent of mineralization and properties of the mineral crystals examined. In all cases where the proteoglycan synthesis or proteoglycans present were modified to provide fewer or smaller molecules, mineralization was enhanced. Specifically, when proteoglycan synthesis was blocked by treatment with 10(-10) M retinoic acid, extensive mineral deposition occurred on a matrix devoid of both proteoglycans and cartilage nodules. The crystals, which formed rapidly, were relatively large in size based on analysis by X-ray diffraction or FT-1R microspectroscopy, and were more abundant than in controls. When 2.5 or 5 mM xylosides were used to cause the synthesis of smaller proteoglycans, the extent of mineral accretion was also increased relative to controls; however, the matrix was less affected, and the extent of mineral deposition and the size of the crystals were not as markedly altered as in the case of retinoic acid. Modification of existing proteoglycans by either chondroinase ABC or hyaluronidase treatment similarly resulted in increased mineral accretion (based on 45Ca uptake or total Ca uptake) relative to cultures in which the proteoglycan content was not manipulated. Crystals were more abundant and larger than in control mineralizing cultures. In contrast, when proteoglycan degradation by metalloproteases was inhibited by metal chelation with o-phenanthroline, the Ca accretion at early time points was increased, but as mineralization progressed, Ca accumulation decreased. These data provide evidence that in this culture system, proteoglycans are inhibitors of mineralization.

Animals↗

Local delivery of an amino bisphosphonate prevents the resorptive phase of alveolar bone following mucoperiosteal flap surgery in rats.

Mucoperiosteal flaps are used to access the bone and root surface in a wide range of periodontal procedures and in implant surgery. We have demonstrated that the mucoperiosteal surgical flap of the rat mandible produces a transient burst of alveolar bone resorption similar to the clinical observations in humans. This resorptive activity, when coupled with local irritation factors, may cause confined alveolar bone loss. Recently, we have demonstrated that an amino bisphosphonate, which is used in preventing systemic bone resorption in osteoporosis and other bone diseases, reduces alveolar bone resorption in the rat model when administered systemically. In this study we evaluated the effect of local delivery of the amino bisphosphonate on bone resorption associated with mucoperiosteal flaps. Following mucoperiosteal flap elevation in the premolar and molar region of the rat mandible, a surgical pellet soaked with amino bisphosphonate was locally applied on the exposed bone surface and covered by flap. The results show that local delivery of amino bisphosphonate reduces significantly alveolar bone resorption activated by mucoperiosteal flap surgery. This study suggests that local application of amino bisphosphonate can be used as an adjunct in therapy for reducing bone resorption following surgery.

Administration, Topical↗

The mechanism of beta-glycerophosphate action in mineralizing chick limb-bud mesenchymal cell cultures.

Differentiating chick limb-bud mesenchymal cells plated in micromass culture form a cartilage matrix that can be mineralized in the presence of 4 mM inorganic phosphate (Pi), and 1 mM calcium. Previous studies showed that when beta-glycerophosphate (beta GP) is used in place of Pi, the mineral crystals formed are larger and differ in distribution. The present study shows that the difference in distribution is not associated with alterations in cell proliferation, protein synthesis, or with collagen, proteoglycan core protein, or alkaline phosphatase gene expression. Cultures with 2.5, 5, and 10 mM beta GP did show different levels of alkaline phosphatase activity, and in the presence of low (0.3 mM) Ca had different Pi contents (4, 6 and 9 mM, respectively), indicating that the increase in CaxP product may in part be responsible for the altered pattern of mineralization. However, cultures with beta GP in which alkaline phosphatase activity was inhibited with levamisole still had an altered mineral distribution as revealed by Fourier transform-infrared (FT-IR) microspectroscopy. The presence of a casein kinase II-like activity in the mineralizing cultures, the ability of specific inhibitors of this enzyme to block mineralization, and the known ability of beta GP to block phosphoprotein phosphatase activity suggests that altered patterns of matrix protein phosphorylation may influence mineral deposition in these cultures.

Alkaline Phosphatase↗

Viable cells are a requirement for in vitro cartilage calcification.

It is a common belief that chondrocyte death must precede calcification in the growth plate. To challenge this dogma, cell devitalization was induced in an in vitro model that mimics in situ cartilage calcification. Chick limb-bud mesenchymal cells, plated in micromass culture, differentiate to form a cartilaginous matrix which mineralizes in the presence of inorganic or organic phosphate. The mineral formed resembles physiologic mineral in crystal size, composition, and distribution. Killing cells by water lysis, ethanol fixation, freeze-thawing, trypsinization, or impairing their function by oligomycin treatment prior to the time at which mineralization commenced, prevented mineral deposition. In contrast, devitalizing cells by any of these techniques after mineralization commenced resulted in dystrophic calcification (excessive, randomly distributed mineral of larger than physiologic crystal size). Based on analyses of 45Ca uptake, FT-IR microscopy, X-ray diffraction, and transmission electron microscopy, it is concluded that the presence of viable cells is obligatory for physiologic cartilage calcification in the differentiating chick limb-bud mesenchymal cell culture system.

Animals↗

The effect of bisphosphonate on alveolar bone resorption following mucoperiosteal flap surgery in the mandible of rats.

Following elevation of a full thickness flap a transient burst of regional remodeling occurs. This phenomenon is termed in orthopedic surgery as regional accelerated phenomenon (RAP), beginning with accelerated resorption activity followed by a slow process of bone regeneration. Recently we have demonstrated that a mucoperiosteal surgical flap of rat mandible is producing a typical RAP process. Bisphosphonates are synthetic compounds that are taken up preferentially by the skeleton and suppress osteoclast-mediated bone resorption by a mechanism that is not yet fully understood. Amino bisphosphonate has been shown to inhibit active bone resorption without interfering with bone formation. In this study we evaluated the effect of amino bisphosphonate on bone resorption associated with a mucoperiosteal flap used as a resorptive model. We compared the effect of amino bisphosphonate in rats using IV administration with topical application at 3 dose levels. The results show that topical application of all 3 doses (0.15, 0.75, and 1.5 mg/ml) had no inhibiting effect on bone resorption after surgery, while IV administration at 0.5 mg/kg body weight significantly reduced the bone resorption. Interestingly, in the non-operated side, amino bisphosphonate increased mineral density.

Administration, Topical↗

Adenosine 5'-triphosphate promotes mineralization in differentiating chick limb-bud mesenchymal cell cultures.

When chick limb-bud mesenchymal cells are plated in micromass culture, they differentiate to form a mineralizable cartilage matrix. Previous studies have demonstrated that, when the total inorganic phosphate concentration of the medium is adjusted to 3-4 mM by adding inorganic phosphate to the basal medium, the mineralized matrix formed resembles that of chick calcified cartilage in ovo. When the high-energy phosphates adenosine 5'-triphosphate (ATP) or creatine phosphate are used as supplements in place of inorganic phosphate, the mineralized matrix as analyzed by electron microscopy and Fourier transform infrared microscopy is also similar to that in ovo. This is in marked contrast to the mineralized matrix formed in the presence of 2.5-5 mM beta-glycerophosphate, where mineral deposition is random and mineral crystal sizes in general are larger. This is also in contrast to the known ability of ATP to inhibit mineral deposition in solution in the absence of cells. In the differentiating mesenchymal cell culture system, ATP does not alter the rate of cell proliferation (DNA content), the rate of matrix synthesis (3H-leucine uptake), the mean crystallite length, or the rate of mineral deposition (45Ca uptake) when contrasted with cultures supplemented with inorganic phosphate. However, ATP does increase the mineral to matrix ratio, especially around the edge of the culture, where a type I collagen matrix is presented. It is suggested that ATP promotes mineral deposition by providing a high-energy phosphate source, which may be used to phosphorylate extracellular matrix proteins and to regulate calcium flux through cell membranes.

Adenosine Triphosphate↗

Regional accelerated phenomenon in the mandible following mucoperiosteal flap surgery.

Striking remodeling activity occurs adjacent to the site of injury in orthopedic surgery. This reaction has been described as regional accelerated phenomenon (RAP), as it speeds up the healing stage. The phenomenon is a transient burst of localized remodeling process following surgical wounding of cortical bone. We explored whether RAP occurs following mucoperiosteal flap surgery in the jaw bone. Mucoperiosteal flaps were performed on 60 Wistar rats, either only on the buccal aspect or both on buccal and lingual aspects of the mandible. The surgical procedure lasted an average of 30 seconds and the flap was readapted without sutures. The rats were sacrificed at 3, 7, 10, 14, 21, and 120 days. High resolution x-ray microradiography of 1 to 1.5 mm thick ground sections between premolar and molar regions of the mandible were analyzed and revealed large areas of radiolucency which correlated to massive resorption of the alveolar bone, as well as areas in the bone proper. The RAP was observed as early as 10 days in the treated side group. Striking resorption of the cortical bone, both on the surface and the bone proper, occurred on the periodontal aspect of the crestal bone leading to widening of the periodontal ligament space, where a mucoperiosteal flap was performed on the buccal aspect. The resorption was more prominent when a mucoperiosteal flap was performed both on the lingual and buccal aspect. The alveolar bone recovered almost to control levels 120 days after surgery.(ABSTRACT TRUNCATED AT 250 WORDS)

Alveolar Bone Loss↗

Technetium 99m-MDP scintigraphy of patients undergoing implant prosthetic procedures: a follow-up study.

The clinical evaluation of peri-implant tissue is mainly based on x-rays. In recent years, radioisotope scintigraphy using Tc-99m-MDP (technetium-99m-methylene diphosphonate) proved to be a useful and reliable clinical method for measuring increased metabolic activity at specific sites of the skeletal tissue. Twenty-six (26) patients (26 to 75 years) who were diagnosed for implant prosthetic treatment modality were randomly chosen to participate in this study. Each patient was injected with 20 mCi of Tc-99m-MDP was scanned 2 hours later by gamma-camera for isotope uptake levels in the jaws. Each patient was scanned 1 to 4 times, starting 2 weeks after implant surgery and up to the 40th week postsurgery. Blade and screw type implants using the non-submerged, one-stage technique were placed. Therefore, the prosthetic reconstruction was initiated 2 weeks after implant surgery. In order to compare the different scans we formulated a relative "bone scan index" (BSI). The results of 62 scans were plotted as BSI against time after implant surgery. A mathematical regression analysis of the BSI was also performed. At the initial stage of 2 to 3 weeks after surgery the BSI is high and then gradually declines. While BSI of the implants in the mandible reaches baseline levels after 12 weeks, the BSI in the maxilla reaches baseline only after 20 weeks. On the other hand, there was no difference in BSI with regard to blade or screw type implant. Here we were able to demonstrate that the process is independent of implant modality and depends on the specific metabolic properties of the jaw.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

FT-IR microscopic mappings of early mineralization in chick limb bud mesenchymal cell cultures.

Chick limb bud mesenchymal cells differentiate into chondrocytes and form a cartilaginous matrix in culture. In this study, the mineral formed in different areas within cultures supplemented with 4 mM inorganic phosphate, or 2.5, 5.0, and 10 mM beta-glycerophosphate (beta GP), was characterized by Fourier-transform infrared (FT-IR) microscopy. The relative mineral-to-matrix ratios, and distribution of crystal sizes at specific locations throughout the matrix were measured from day 14 to day 30. The only mineral phase detected was a poorly crystalline apatite. Cultures receiving 4 mM inorganic phosphate had smaller crystals which were less randomly distributed around the cartilage nodules than those in the beta GP-treated cultures. beta GP-induced mineral consisted of larger, more perfect apatite crystals. In cultures receiving 5 or 10 mM beta GP, the relative mineral-to-matrix ratios (calculated from the integrated intensities of the phosphate and amide I bands, respectively) were higher than in the cultures with 4 mM inorganic phosphate or in the in vivo calcified chick cartilage.

Animals↗