ObjectivesNovel information on apartheid health conditions may be obtained through the study of r... more ObjectivesNovel information on apartheid health conditions may be obtained through the study of recent skeletal collections. Using a backscattered scanning electron microscopy (BSE‐SEM) approach, this study aims to produce bone quality and tissue mineralization data for an understudied South African population from the Western Cape province.MethodsUsing BSE‐SEM imaging, cortical porosity (Ct.Po), osteocyte lacunar density (Ot.Lc.Dn), and the degree of tissue mineralization were quantified in mid‐thoracic ribs from the Kirsten Skeletal Collection. Individuals ( nfemale = 75, nmale = 68, and mean age = 46.3 years) were predominantly from the South Africa Colored (SAC) population group ( nSAC = 103, 72%). Full cross‐sectional images of each rib were manually stitched together in Adobe Photoshop. Photomontages were imported into MATALB (Mathworks, Natick, MA) for image processing and analysis. Age‐related changes in histomorphometric parameters and sex differences were examined using co...
American journal of human biology : the official journal of the Human Biology Council, Jan 7, 2018
The purpose of this study was to provide bone histomorphometric reference data for South Africans... more The purpose of this study was to provide bone histomorphometric reference data for South Africans of the Western Cape who likely dealt with health issues under the apartheid regime. The 206 adult individuals (female = 75,male = 131, mean = 47.9 ± 15.8 years) from the Kirsten Skeletal Collection, U. Stellenbosch, lived in the Cape Town metropole from the late 1960s to the mid-1990s. To study age-related changes in cortical and trabecular bone microstructure, photomontages of mid-thoracic rib cross-sections were quantitatively examined. Variables include relative cortical area (Rt.Ct.Ar), osteon population density (OPD), osteon area (On.Ar), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). All cortical variables demonstrated significant relationships with age in both sexes, with women showing stronger overall age associations. Peak bone mass was compromised in some men, possibly reflecting poor nutritional quality an...
Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This target... more Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This targeting ability has been exploited to design and prepare bisphosphonate conjugate prodrugs to deliver a wide variety of drug molecules selectively to bones. It is important that conjugates be stable in the blood stream and that conjugate that is not taken up by bone is eliminated rapidly. The prodrugs should release active drug at a rate appropriate so as to provide efficacy. Radiolabelling is the best method to quantify and evaluate pharmacokinetics, tissue distribution, bone uptake and release of the active drug(s). Recent reports have described bisphosphonate conjugates derived from the antiresorptive drug, alendronic acid and anabolic prostanoid drugs that effectively deliver prostaglandins and prostaglandin EP4 receptor agonists to bone and show enhanced anabolic efficacy and tolerability compared to the drugs alone. These conjugate drugs can be dosed infrequently (weekly or bimonthly) ...
SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : RP 16327 / INIST-CN... more SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : RP 16327 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc
We combined dual-photon absorptiometry, iliac crest histomorphometry, and backscattered electrons... more We combined dual-photon absorptiometry, iliac crest histomorphometry, and backscattered electrons analysis to characterize bone mineralization effects of a spaceflight on young monkeys. Two 4- to 5-kg male rhesus monkeys (Macaca mulatta) were flown during a 11.5-day spaceflight that took place onboard Cosmos 2229 biosatellite (Bion 10). Vivarium (n = 4) and Earth-based chair (n = 4) control situations were studied for comparison. Flight monkeys exhibited lower values of iliac cancellous bone volume, associated with nonsignificantly thinner trabeculae. Bone mineralization rate and the proportion of trabecular bone surface involved in mineralization processes were found markedly reduced after spaceflight. Analysis of embedded sections by backscattered electrons imaging showed a nonsignificant shift to lower mineralization in the flight biopsies vs. postflight mock-up biopsies. These results were in accordance with dual-photon absorptiometry evaluations showing a tendency for decreased...
Phosphorus (P) is an important and limiting element for life. One strategy for storing ortho phos... more Phosphorus (P) is an important and limiting element for life. One strategy for storing ortho phosphates (Pi) is polymerization. Polymerized Pi's (polyphosphates: (PO3-)n: polyPs) serve as a Pi bank, as well as a catiion chelator, energy source, & regulator of responses to stresses in the stationary phase of culture growth and development1. PolyP biochemistry has been investigated in yeasts, bacteria
The benefit of glucosamine (GlcN) in bone and joint disorders remains controversial. N-acetylatio... more The benefit of glucosamine (GlcN) in bone and joint disorders remains controversial. N-acetylation and other N-acylations of GlcN alter its biological properties fundamentally. We have shown previously that N-butyryl glucosamine (GlcNBu) preserved strikingly the subchondral bone structure in a destructive arthritis rat model. Here, we examine whether GlcNBu preserves bone in the ovariectomized (OVX) rat, a model for postmenopausal osteoporosis. Rats were randomized into 4 groups: group 1, sham OVX glucose (Glc) fed; group 2, sham OVX GlcNBu fed; group 3, OVX Glc fed; and group 4, OVX GlcNBu fed. A single, oral, 200-mg/kg dose of GlcNBu or Glc was administered daily for 6 months. Bone mineral content (BMC) and bone mineral density, and biomechanical properties of the femurs and spines were determined by standardized techniques. Two-way analysis of variance with a Bonferroni post hoc test was used for statistical analysis. Ovariectomy in group 3 resulted either in significant or highly significant effects in a number of the tests. For spinal BMCs the interaction between GlcNBu and OVX was significant. For the femurs, this interaction was also seen in energy to failure, and ultimate displacement and ultimate strain tests. In general, ovariectomy was necessary to show significant preventive effects of GlcNBu on mineral content and some biomechanical properties. We conclude that GlcNBu feeding in the OVX rat preserves bone mineral and some biomechanical properties. Translationally, GlcNBu can be positioned between nutriceuticals and pharmaceuticals for the prevention and treatment of osteoporosis. Advantages include low production costs and a favorable safety profile.
Cartilagenous tissue with mineralized and nonmineralized layers was generated in vitro using bovi... more Cartilagenous tissue with mineralized and nonmineralized layers was generated in vitro using bovine chondrocytes isolated from the deep zone of articular cartilage. Mineralization was induced by addition of either /3-glycerophosphate (/3-GP), phosphoethanolamine (PEA), or adenosine triphosphate (ATP). As this tissue might be suitable for use in joint resurfacing, the mineral of the calcifled layer was characterized and compared to that present in the in vivo mineralized zone of bovine articular cartilage. Von Kossa staining demonstrated the presence of mineralization in the lower half of the tissue. The calcium content in the tissue varied from 4.9% to 7.8% of dry weight. Electron diffraction demonstrated a pattern consistent with hydroxyapatite. Brightfleld transmission electron microscopy showed that the crystals were acicular and when measured under electron diffraction dark fleld imaging were 16.6 ± 3.8 (/3-GP), 16.4 ± 3.8 nm (ATP), and 17.0 ± 6.3 nm (PEA) in length. The crystals were similar in size (16.0 ± 5.5 nm) and appearance to the crystals in the in vivo calcifled cartilage. This data suggests that the mineralization that occurs in vitro is similar to the in vivo cartilage.
Objective: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content i... more Objective: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content in cartilage accelerates the progression of spontaneous osteoarthritis (OA) in the Hartley guinea pig (HGP) model. Methods: Twenty-eight male, 3-month-old HGPs were used. Eight were left untreated as a baseline control group and sacrificed at 3 months of age (n ¼ 4) and 9 months of age (n ¼ 4; age-matched controls). The other 20 HGPs received intra-articular knee injections in the right knee whereas the left knees acted as contra-lateral non-injected controls. Injections consisted of 100 ml phosphate buffered saline (PBS; n ¼ 10) or PBSþ2.0 M D-(À)-Ribose (n ¼ 10). Injections were given once weekly for 24 weeks. At the end of the treatment period, the tibiae were fixed with formalin, scanned with microCT for subchondral bone mineral density, and then histological slides were prepared, stained with Safranin-O with Fast Green counter stain and scored using the OARSI-HISTOgp scheme. Cartilage pentosidine (established biomarker for AGEs) content, collagen content (% dry mass), glucosaminoglycan GAG-to-collagen ratio (mg/mg), GAG-to-DNA ratio and DNA-to-collagen ratio were measured. Results: Pentosidine content increased greatly due to PBS þ Ribose injection (P < 0.0001) and reached levels found in cartilage from 80-year-old humans. Surprisingly, mean OARSI-HISTOgp scores for both the injected and contra-lateral controls in the PBS þ Ribose group were not detectably different, nor were they different from the mean score for the age-matched control group. Conclusion: AGEs accumulation due to intra-articular ribose-containing injections in the HGP model of spontaneous knee OA did not enhance disease progression.
Crystal-associated nephropathy in patients with brushite nephrolithiasis. Background. We have bio... more Crystal-associated nephropathy in patients with brushite nephrolithiasis. Background. We have biopsied the renal cortex and papillae of patients who form brushite renal stones asking if this unusual stone type is associated with specific tissue changes. We contrasted these with biopsies of 15 calcium oxalate stone formers, three stone formers with intestinal bypass, and four normal subjects. Methods. We studied all ten brushite stone formers treated with percutaneous nephrolithotomy (PNL) during the past 3 years using digital video imaging of renal papillae, and obtained cortical and papillary biopsies. Biopsies were analyzed by light and electron microscopy, microinfrared spectroscopy, and electron diffraction. Results. Apatite crystals plugged scattered terminal collecting ducts whose cells were injured or dead, and surrounding interstitium inflamed and fibrotic. White papillary deposits of interstitial apatite particles, so called Randall's plaque, were also present. Glomerular changes and cortical tubular atrophy and interstitial fibrosis were moderate to severe. Conclusion. Brushite stone formers combine the interstitial plaque of calcium oxalate stone formers with the collecting duct apatite plugs found in stone formers with intestinal bypass. Collecting duct injury and interstitial fibrosis are severe. Prominent cortical fibrosis, tubule atrophy, and glomerular pathology seem secondary to the collecting duct plugging. We believe crystallization obstructs and destroys terminal collecting duct segments thereby damaging nephrons, perhaps via intranephronal obstruction, and producing a hitherto unrecognized renal disease.
In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell ... more In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell culture system that resulted in the formation of NP-like tissue in vitro. NP cells were isolated from sheep lumbar spines and grown on filter inserts (Millicell CM). Histological examination showed that the cells accumulated extracellular matrix and formed a continuous layer of NP-like tissue. The accumulation of sulfated proteoglycans in the NP-like tissue continued up to 10 weeks and this was paralleled by an increase in tissue thickness and dry weight. DNA content remained stable during the first 4 weeks but then decreased over time. The amount of DNA, glycosaminoglycan (GAG) and collagen per mg dry weight of the tissue generated after 10 weeks in culture were 1.25+/-0.02, 301.6+/-27.7 and 411+/-65 microg, respectively, compared with 1.04+/-0.08, 320.6+/-21.2 and 399+/-4.4 microg (mean +/- SEM) for the in vivo tissue. There was no significant difference between in vitro and in vivo tissue. The cells in culture synthesized large proteoglycans (kav = 0.26+/-0.03, mean +/- S.D.) which were similar in size to those synthesized by cells in NP tissue in ex vivo culture (kav = 0.22+/-0.02, mean +/- S.D.) as determined by Sepharose CL-2B column chromatography. The in vitro generated tissue contained type II collagen as demonstrated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining as well as Western blot analysis. NP cells grown on filters generate tissue similar in composition to the in vivo tissue, for the characteristics examined to date, and should be a suitable model to use to study NP metabolism and extracellular matrix turnover.
Management of soft tissue sarcoma involves multimodality treatment, including surgery and radioth... more Management of soft tissue sarcoma involves multimodality treatment, including surgery and radiotherapy. Pathologic fracture of the femur after such treatment in the thigh is one serious, late complication and nonunion rates of 80-90% are reported. We hypothesize that the combination of radiotherapy and periosteal stripping (during tumor resection) leads to greater impairment of the fracture repair process than either intervention alone. Female Wistar retired breeder rats were randomized into four treatment groups (control, radiotherapy, surgery, and combination of radiotherapy and surgery) and three end-points (21, 28, and 35 days post-fracture). Designated animals first underwent radiotherapy, followed by surgical stripping of the periosteum 3 weeks later and femoral fracture with fixation after another 3 weeks. Animals were sacrificed and fractures examined using microCT and histomorphometry. Simple transverse or short oblique femoral fractures were produced. By 35 days, control animals formed unions, periosteum-stripped animals formed hypertrophic non-unions and irradiated animals formed atrophic non-unions. Histomorphometry revealed an absence of chondroid and osteoid production in animals undergoing radiotherapy. The relative contribution of periosteal stripping to occurrence of non-union was statistically insignificant. Radiation prior to fracture reliably resulted in atrophic non-union in our model. The contribution of periosteal stripping was negligible.
We have bred a strain of rats to maximize urine (u) calcium (Ca) excretion and model hypercalciur... more We have bred a strain of rats to maximize urine (u) calcium (Ca) excretion and model hypercalciuric nephrolithiasis. These genetic hypercalciuric stone-forming (GHS) rats excrete more uCa than control Sprague-Dawley rats, uniformly form kidney stones, and similar to patients, demonstrate lower bone mineral density. Clinically, thiazide diuretics reduce uCa and prevent stone formation; however, whether they benefit bone is not clear. We used GHS rats to test the hypothesis that the thiazide diuretic chlorthalidone (CTD) would have a favorable effect on bone density and quality. Twenty GHS rats received a fixed amount of a 1.2% Ca diet, and half also were fed CTD (4 to 5 mg/kg/d). Rats fed CTD had a marked reduction in uCa. The axial and appendicular skeletons were studied. An increase in trabecular mineralization was observed with CTD compared with controls. CTD also improved the architecture of trabecular bone. Using micro-computed tomography (mCT), trabecular bone volume (BV/TV), trabecular thickness, and trabecular number were increased with CTD. A significant increase in trabecular thickness with CTD was confirmed by static histomorphometry. CTD also improved the connectivity of trabecular bone. Significant improvements in vertebral strength and stiffness were measured by vertebral compression. Conversely, a slight loss of bending strength was detected in the femoral diaphysis with CTD. Thus results obtained in hypercalciuric rats suggest that CTD can favorably influence vertebral fracture risk. CTD did not alter formation parameters, suggesting that the improved vertebral bone strength was due to decreased bone resorption and retention of bone structure.
Kidney stone patients often have a decrease in BMD. It is unclear if reduced BMD is caused by a p... more Kidney stone patients often have a decrease in BMD. It is unclear if reduced BMD is caused by a primary disorder of bone or dietary factors. To study the independent effects of hypercalciuria on bone, we used genetic hypercalciuric stone-forming (GHS) rats. GHS and control (Ctl) rats were fed a low Ca (0.02% Ca, LCD) or a high Ca (1.2% Ca, HCD) diet for 6 wk in metabolic cages. All comparisons are to Ctl rats. Urine Ca was greater in the GHS rats on both diets. GHS fed HCD had reduced cortical (humerus) and trabecular (L 1-L 5 vertebrae) BMD, whereas GHS rats fed LCD had a reduction in BMD similar to Ctl. GHS rats fed HCD had a decrease in trabecular volume and thickness, whereas LCD led to a ;20-fold increase in both osteoid surface and volume. GHS rats fed HCD had no change in vertebral strength (failure stress), ductibility (failure strain), stiffness (modulus), or toughness, whereas in the humerus, there was reduced ductibility and toughness and an increase in modulus, indicating that the defect in mechanical properties is mainly manifested in cortical, rather than trabecular, bone. GHS rat cortical bone is more mineralized than trabecular bone and LCD led to a decrease in the mineralization profile. Thus, the GHS rats, fed an ample Ca diet, have reduced BMD with reduced trabecular volume, mineralized volume, and thickness, and their bones are more brittle and fracture prone, indicating that GHS rats have an intrinsic disorder of bone that is not secondary to diet.
Resolution enhanced FT-IR spectroscopy shows that non-apatitic environments of phosphate and carb... more Resolution enhanced FT-IR spectroscopy shows that non-apatitic environments of phosphate and carbonate ions occur in bone mineral. The spectroscopic characteristics of these environments and their chemical and biological properties are reviewed. The potential effectiveness of FT-IR for the detailed study of bone mineral changes in bone disease is shown by analysis of several samples.
ObjectivesNovel information on apartheid health conditions may be obtained through the study of r... more ObjectivesNovel information on apartheid health conditions may be obtained through the study of recent skeletal collections. Using a backscattered scanning electron microscopy (BSE‐SEM) approach, this study aims to produce bone quality and tissue mineralization data for an understudied South African population from the Western Cape province.MethodsUsing BSE‐SEM imaging, cortical porosity (Ct.Po), osteocyte lacunar density (Ot.Lc.Dn), and the degree of tissue mineralization were quantified in mid‐thoracic ribs from the Kirsten Skeletal Collection. Individuals ( nfemale = 75, nmale = 68, and mean age = 46.3 years) were predominantly from the South Africa Colored (SAC) population group ( nSAC = 103, 72%). Full cross‐sectional images of each rib were manually stitched together in Adobe Photoshop. Photomontages were imported into MATALB (Mathworks, Natick, MA) for image processing and analysis. Age‐related changes in histomorphometric parameters and sex differences were examined using co...
American journal of human biology : the official journal of the Human Biology Council, Jan 7, 2018
The purpose of this study was to provide bone histomorphometric reference data for South Africans... more The purpose of this study was to provide bone histomorphometric reference data for South Africans of the Western Cape who likely dealt with health issues under the apartheid regime. The 206 adult individuals (female = 75,male = 131, mean = 47.9 ± 15.8 years) from the Kirsten Skeletal Collection, U. Stellenbosch, lived in the Cape Town metropole from the late 1960s to the mid-1990s. To study age-related changes in cortical and trabecular bone microstructure, photomontages of mid-thoracic rib cross-sections were quantitatively examined. Variables include relative cortical area (Rt.Ct.Ar), osteon population density (OPD), osteon area (On.Ar), bone volume fraction (BV/TV), trabecular number (Tb.N), trabecular thickness (Tb.Th), and trabecular spacing (Tb.Sp). All cortical variables demonstrated significant relationships with age in both sexes, with women showing stronger overall age associations. Peak bone mass was compromised in some men, possibly reflecting poor nutritional quality an...
Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This target... more Bisphosphonates target and bind avidly to the mineral (hydroxyapatite) found in bone. This targeting ability has been exploited to design and prepare bisphosphonate conjugate prodrugs to deliver a wide variety of drug molecules selectively to bones. It is important that conjugates be stable in the blood stream and that conjugate that is not taken up by bone is eliminated rapidly. The prodrugs should release active drug at a rate appropriate so as to provide efficacy. Radiolabelling is the best method to quantify and evaluate pharmacokinetics, tissue distribution, bone uptake and release of the active drug(s). Recent reports have described bisphosphonate conjugates derived from the antiresorptive drug, alendronic acid and anabolic prostanoid drugs that effectively deliver prostaglandins and prostaglandin EP4 receptor agonists to bone and show enhanced anabolic efficacy and tolerability compared to the drugs alone. These conjugate drugs can be dosed infrequently (weekly or bimonthly) ...
SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : RP 16327 / INIST-CN... more SIGLEAvailable from INIST (FR), Document Supply Service, under shelf-number : RP 16327 / INIST-CNRS - Institut de l'Information Scientifique et TechniqueFRFranc
We combined dual-photon absorptiometry, iliac crest histomorphometry, and backscattered electrons... more We combined dual-photon absorptiometry, iliac crest histomorphometry, and backscattered electrons analysis to characterize bone mineralization effects of a spaceflight on young monkeys. Two 4- to 5-kg male rhesus monkeys (Macaca mulatta) were flown during a 11.5-day spaceflight that took place onboard Cosmos 2229 biosatellite (Bion 10). Vivarium (n = 4) and Earth-based chair (n = 4) control situations were studied for comparison. Flight monkeys exhibited lower values of iliac cancellous bone volume, associated with nonsignificantly thinner trabeculae. Bone mineralization rate and the proportion of trabecular bone surface involved in mineralization processes were found markedly reduced after spaceflight. Analysis of embedded sections by backscattered electrons imaging showed a nonsignificant shift to lower mineralization in the flight biopsies vs. postflight mock-up biopsies. These results were in accordance with dual-photon absorptiometry evaluations showing a tendency for decreased...
Phosphorus (P) is an important and limiting element for life. One strategy for storing ortho phos... more Phosphorus (P) is an important and limiting element for life. One strategy for storing ortho phosphates (Pi) is polymerization. Polymerized Pi's (polyphosphates: (PO3-)n: polyPs) serve as a Pi bank, as well as a catiion chelator, energy source, & regulator of responses to stresses in the stationary phase of culture growth and development1. PolyP biochemistry has been investigated in yeasts, bacteria
The benefit of glucosamine (GlcN) in bone and joint disorders remains controversial. N-acetylatio... more The benefit of glucosamine (GlcN) in bone and joint disorders remains controversial. N-acetylation and other N-acylations of GlcN alter its biological properties fundamentally. We have shown previously that N-butyryl glucosamine (GlcNBu) preserved strikingly the subchondral bone structure in a destructive arthritis rat model. Here, we examine whether GlcNBu preserves bone in the ovariectomized (OVX) rat, a model for postmenopausal osteoporosis. Rats were randomized into 4 groups: group 1, sham OVX glucose (Glc) fed; group 2, sham OVX GlcNBu fed; group 3, OVX Glc fed; and group 4, OVX GlcNBu fed. A single, oral, 200-mg/kg dose of GlcNBu or Glc was administered daily for 6 months. Bone mineral content (BMC) and bone mineral density, and biomechanical properties of the femurs and spines were determined by standardized techniques. Two-way analysis of variance with a Bonferroni post hoc test was used for statistical analysis. Ovariectomy in group 3 resulted either in significant or highly significant effects in a number of the tests. For spinal BMCs the interaction between GlcNBu and OVX was significant. For the femurs, this interaction was also seen in energy to failure, and ultimate displacement and ultimate strain tests. In general, ovariectomy was necessary to show significant preventive effects of GlcNBu on mineral content and some biomechanical properties. We conclude that GlcNBu feeding in the OVX rat preserves bone mineral and some biomechanical properties. Translationally, GlcNBu can be positioned between nutriceuticals and pharmaceuticals for the prevention and treatment of osteoporosis. Advantages include low production costs and a favorable safety profile.
Cartilagenous tissue with mineralized and nonmineralized layers was generated in vitro using bovi... more Cartilagenous tissue with mineralized and nonmineralized layers was generated in vitro using bovine chondrocytes isolated from the deep zone of articular cartilage. Mineralization was induced by addition of either /3-glycerophosphate (/3-GP), phosphoethanolamine (PEA), or adenosine triphosphate (ATP). As this tissue might be suitable for use in joint resurfacing, the mineral of the calcifled layer was characterized and compared to that present in the in vivo mineralized zone of bovine articular cartilage. Von Kossa staining demonstrated the presence of mineralization in the lower half of the tissue. The calcium content in the tissue varied from 4.9% to 7.8% of dry weight. Electron diffraction demonstrated a pattern consistent with hydroxyapatite. Brightfleld transmission electron microscopy showed that the crystals were acicular and when measured under electron diffraction dark fleld imaging were 16.6 ± 3.8 (/3-GP), 16.4 ± 3.8 nm (ATP), and 17.0 ± 6.3 nm (PEA) in length. The crystals were similar in size (16.0 ± 5.5 nm) and appearance to the crystals in the in vivo calcifled cartilage. This data suggests that the mineralization that occurs in vitro is similar to the in vivo cartilage.
Objective: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content i... more Objective: To test the hypothesis that heightened advanced glycation endproducts (AGEs) content in cartilage accelerates the progression of spontaneous osteoarthritis (OA) in the Hartley guinea pig (HGP) model. Methods: Twenty-eight male, 3-month-old HGPs were used. Eight were left untreated as a baseline control group and sacrificed at 3 months of age (n ¼ 4) and 9 months of age (n ¼ 4; age-matched controls). The other 20 HGPs received intra-articular knee injections in the right knee whereas the left knees acted as contra-lateral non-injected controls. Injections consisted of 100 ml phosphate buffered saline (PBS; n ¼ 10) or PBSþ2.0 M D-(À)-Ribose (n ¼ 10). Injections were given once weekly for 24 weeks. At the end of the treatment period, the tibiae were fixed with formalin, scanned with microCT for subchondral bone mineral density, and then histological slides were prepared, stained with Safranin-O with Fast Green counter stain and scored using the OARSI-HISTOgp scheme. Cartilage pentosidine (established biomarker for AGEs) content, collagen content (% dry mass), glucosaminoglycan GAG-to-collagen ratio (mg/mg), GAG-to-DNA ratio and DNA-to-collagen ratio were measured. Results: Pentosidine content increased greatly due to PBS þ Ribose injection (P < 0.0001) and reached levels found in cartilage from 80-year-old humans. Surprisingly, mean OARSI-HISTOgp scores for both the injected and contra-lateral controls in the PBS þ Ribose group were not detectably different, nor were they different from the mean score for the age-matched control group. Conclusion: AGEs accumulation due to intra-articular ribose-containing injections in the HGP model of spontaneous knee OA did not enhance disease progression.
Crystal-associated nephropathy in patients with brushite nephrolithiasis. Background. We have bio... more Crystal-associated nephropathy in patients with brushite nephrolithiasis. Background. We have biopsied the renal cortex and papillae of patients who form brushite renal stones asking if this unusual stone type is associated with specific tissue changes. We contrasted these with biopsies of 15 calcium oxalate stone formers, three stone formers with intestinal bypass, and four normal subjects. Methods. We studied all ten brushite stone formers treated with percutaneous nephrolithotomy (PNL) during the past 3 years using digital video imaging of renal papillae, and obtained cortical and papillary biopsies. Biopsies were analyzed by light and electron microscopy, microinfrared spectroscopy, and electron diffraction. Results. Apatite crystals plugged scattered terminal collecting ducts whose cells were injured or dead, and surrounding interstitium inflamed and fibrotic. White papillary deposits of interstitial apatite particles, so called Randall's plaque, were also present. Glomerular changes and cortical tubular atrophy and interstitial fibrosis were moderate to severe. Conclusion. Brushite stone formers combine the interstitial plaque of calcium oxalate stone formers with the collecting duct apatite plugs found in stone formers with intestinal bypass. Collecting duct injury and interstitial fibrosis are severe. Prominent cortical fibrosis, tubule atrophy, and glomerular pathology seem secondary to the collecting duct plugging. We believe crystallization obstructs and destroys terminal collecting duct segments thereby damaging nephrons, perhaps via intranephronal obstruction, and producing a hitherto unrecognized renal disease.
In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell ... more In order to be able to study the metabolism of nucleus pulposus (NP) tissue, we developed a cell culture system that resulted in the formation of NP-like tissue in vitro. NP cells were isolated from sheep lumbar spines and grown on filter inserts (Millicell CM). Histological examination showed that the cells accumulated extracellular matrix and formed a continuous layer of NP-like tissue. The accumulation of sulfated proteoglycans in the NP-like tissue continued up to 10 weeks and this was paralleled by an increase in tissue thickness and dry weight. DNA content remained stable during the first 4 weeks but then decreased over time. The amount of DNA, glycosaminoglycan (GAG) and collagen per mg dry weight of the tissue generated after 10 weeks in culture were 1.25+/-0.02, 301.6+/-27.7 and 411+/-65 microg, respectively, compared with 1.04+/-0.08, 320.6+/-21.2 and 399+/-4.4 microg (mean +/- SEM) for the in vivo tissue. There was no significant difference between in vitro and in vivo tissue. The cells in culture synthesized large proteoglycans (kav = 0.26+/-0.03, mean +/- S.D.) which were similar in size to those synthesized by cells in NP tissue in ex vivo culture (kav = 0.22+/-0.02, mean +/- S.D.) as determined by Sepharose CL-2B column chromatography. The in vitro generated tissue contained type II collagen as demonstrated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE) and silver staining as well as Western blot analysis. NP cells grown on filters generate tissue similar in composition to the in vivo tissue, for the characteristics examined to date, and should be a suitable model to use to study NP metabolism and extracellular matrix turnover.
Management of soft tissue sarcoma involves multimodality treatment, including surgery and radioth... more Management of soft tissue sarcoma involves multimodality treatment, including surgery and radiotherapy. Pathologic fracture of the femur after such treatment in the thigh is one serious, late complication and nonunion rates of 80-90% are reported. We hypothesize that the combination of radiotherapy and periosteal stripping (during tumor resection) leads to greater impairment of the fracture repair process than either intervention alone. Female Wistar retired breeder rats were randomized into four treatment groups (control, radiotherapy, surgery, and combination of radiotherapy and surgery) and three end-points (21, 28, and 35 days post-fracture). Designated animals first underwent radiotherapy, followed by surgical stripping of the periosteum 3 weeks later and femoral fracture with fixation after another 3 weeks. Animals were sacrificed and fractures examined using microCT and histomorphometry. Simple transverse or short oblique femoral fractures were produced. By 35 days, control animals formed unions, periosteum-stripped animals formed hypertrophic non-unions and irradiated animals formed atrophic non-unions. Histomorphometry revealed an absence of chondroid and osteoid production in animals undergoing radiotherapy. The relative contribution of periosteal stripping to occurrence of non-union was statistically insignificant. Radiation prior to fracture reliably resulted in atrophic non-union in our model. The contribution of periosteal stripping was negligible.
We have bred a strain of rats to maximize urine (u) calcium (Ca) excretion and model hypercalciur... more We have bred a strain of rats to maximize urine (u) calcium (Ca) excretion and model hypercalciuric nephrolithiasis. These genetic hypercalciuric stone-forming (GHS) rats excrete more uCa than control Sprague-Dawley rats, uniformly form kidney stones, and similar to patients, demonstrate lower bone mineral density. Clinically, thiazide diuretics reduce uCa and prevent stone formation; however, whether they benefit bone is not clear. We used GHS rats to test the hypothesis that the thiazide diuretic chlorthalidone (CTD) would have a favorable effect on bone density and quality. Twenty GHS rats received a fixed amount of a 1.2% Ca diet, and half also were fed CTD (4 to 5 mg/kg/d). Rats fed CTD had a marked reduction in uCa. The axial and appendicular skeletons were studied. An increase in trabecular mineralization was observed with CTD compared with controls. CTD also improved the architecture of trabecular bone. Using micro-computed tomography (mCT), trabecular bone volume (BV/TV), trabecular thickness, and trabecular number were increased with CTD. A significant increase in trabecular thickness with CTD was confirmed by static histomorphometry. CTD also improved the connectivity of trabecular bone. Significant improvements in vertebral strength and stiffness were measured by vertebral compression. Conversely, a slight loss of bending strength was detected in the femoral diaphysis with CTD. Thus results obtained in hypercalciuric rats suggest that CTD can favorably influence vertebral fracture risk. CTD did not alter formation parameters, suggesting that the improved vertebral bone strength was due to decreased bone resorption and retention of bone structure.
Kidney stone patients often have a decrease in BMD. It is unclear if reduced BMD is caused by a p... more Kidney stone patients often have a decrease in BMD. It is unclear if reduced BMD is caused by a primary disorder of bone or dietary factors. To study the independent effects of hypercalciuria on bone, we used genetic hypercalciuric stone-forming (GHS) rats. GHS and control (Ctl) rats were fed a low Ca (0.02% Ca, LCD) or a high Ca (1.2% Ca, HCD) diet for 6 wk in metabolic cages. All comparisons are to Ctl rats. Urine Ca was greater in the GHS rats on both diets. GHS fed HCD had reduced cortical (humerus) and trabecular (L 1-L 5 vertebrae) BMD, whereas GHS rats fed LCD had a reduction in BMD similar to Ctl. GHS rats fed HCD had a decrease in trabecular volume and thickness, whereas LCD led to a ;20-fold increase in both osteoid surface and volume. GHS rats fed HCD had no change in vertebral strength (failure stress), ductibility (failure strain), stiffness (modulus), or toughness, whereas in the humerus, there was reduced ductibility and toughness and an increase in modulus, indicating that the defect in mechanical properties is mainly manifested in cortical, rather than trabecular, bone. GHS rat cortical bone is more mineralized than trabecular bone and LCD led to a decrease in the mineralization profile. Thus, the GHS rats, fed an ample Ca diet, have reduced BMD with reduced trabecular volume, mineralized volume, and thickness, and their bones are more brittle and fracture prone, indicating that GHS rats have an intrinsic disorder of bone that is not secondary to diet.
Resolution enhanced FT-IR spectroscopy shows that non-apatitic environments of phosphate and carb... more Resolution enhanced FT-IR spectroscopy shows that non-apatitic environments of phosphate and carbonate ions occur in bone mineral. The spectroscopic characteristics of these environments and their chemical and biological properties are reviewed. The potential effectiveness of FT-IR for the detailed study of bone mineral changes in bone disease is shown by analysis of several samples.
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