Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a polyanhydride delivery vehicle.

Controlled release delivery vehicles for water-soluble osteogenic proteins from demineralized bovine bone matrix were constructed using polyanhydride polymers. The water-soluble proteins were isolated from a 4 M guanidine hydrochloride extract of bone matrix. The water-soluble proteins possessed Chondrogenic Stimulating Activity (CSA) when tested in stage 24 chick limb bud cell cultures, but were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice by themselves. The polyanhydride polymers alone were also incapable of inducing ectopic cartilage or bone. However, when the water-soluble proteins were incorporated into the polymeric delivery vehicle, the combination was capable of inducing cartilage and bone up to 50% of the time. These results demonstrate that it is possible to use polyanhydride polymers as controlled-release delivery vehicles for soluble bioactive factors that interact with a local cell population.

Chondrogenic stimulation of embryonic chick limb mesenchymal cells by factors in bovine and human dentine extracts.

Extracts of bovine or human dentine when incorporated into the medium bathing embryonic chick-limb mesenchymal cells caused the stimulation of chondrogenesis. Such extracts were prepared from demineralized bovine or human dentine by suspension in a 4 M guanidinium hydrochloride (GuHCl) solution. After stepwise dialysis down to distilled water to remove the GuHCl, the protein extracts were separated chromatographically on DEAE resin. The crude and separated extracts from both dentines produced morphological changes in the cultured mesenchymal cells similar to those reported for bovine and human adult bone extracts, as well as metachromatic staining with toluidine blue indicating that they stimulated chondrogenesis. Measurements of [35S]-SO4 incorporation and DNA content in the cultures also indicated that the extracts stimulated chondrogenesis but not cell proliferation. The similarity between silver-stained patterns on sodium dodecyl sulphate polyacrylamide gel electrophoresis of corresponding fractions of the bovine and human dentine suggests that they may contain similar or identical proteins.

Ectopic induction of cartilage and bone by water-soluble proteins from bovine bone using a collagenous delivery vehicle.

A controlled-release delivery vehicle for water-soluble osteogenic proteins from demineralized bone matrix was constructed using purified type I collagen. The water-soluble proteins were isolated from a 4 M GdnHCl extract of bone matrix. Although the water-soluble proteins were capable of inducing cartilage formation in vitro, they were incapable of inducing cartilage or bone in vivo when implanted intramuscularly into mice in the absence of an appropriate delivery vehicle. The collagen-based delivery vehicle alone was also incapable of inducing osteogenesis in vivo. However, when the water-soluble proteins were incorporated into the delivery vehicle, the combination was capable of inducing cartilage and bone 76% of the time. These results demonstrate that it is possible to formulate a controlled-release delivery vehicles for soluble bioactive factors which upon release interact with local responsive cells.

A water-soluble fraction from adult bone stimulates the differentiation of cartilage in explants of embryonic muscle.

A water-soluble fraction of a 4 M guanidine HCl extract of demineralized adult bovine bone stimulated the differentiation of cartilage in explants of minced skeletal muscle from embryonic chick legs; cartilage was also induced by a semipurified protein preparation. Cartilage could be identified in treated cultures at 1 week with muscle from day-9 embryos, not before 2 weeks with muscle from day-12 embryos, and not before 3 weeks with muscle from day-19 embryos. The ability to respond to this water-soluble fraction by exhibiting cartilage differentiation was dose-dependent, but not confined to any particular muscle region of the day-12 embryonic leg. These observations indicate that bone-derived soluble chondroinductive agents act on cells in minced embryonic muscle preparations. The induction of cartilage is dependent upon the accessibility of the responding cells to the agents, on the concentration of inductive agents, and on the developmental age of the responsive tissue.

In vitro chondrogenesis stimulated by extracts of gall bladder epithelium.

Living adult gall bladder epithelium has previously been shown to initiate in vivo ectopic cartilage and/or bone formation. In the present study, we demonstrate that solubilized extracts of gall bladder, which have never been inductive when tested in vivo, do in fact contain a factor that stimulates chondrogenesis in stage 24 chick limb bud mesenchymal cell cultures. These 4 M guanidinium chloride soluble, cold water soluble protein fractions initiate a dose-dependent increase in the amount of cartilage formed following an 8-10 day continuous exposure as evidenced by the presence of greater numbers of chondrocytes when viewed with phase optics, more intense toluidine blue metachromatic staining in fixed plates and elevated 35S-sulfate incorporation into the cell layer (as a maker for proteoglycan biosynthesis). The factor(s) responsible for this activity binds to DEAE anion-exchange resins, is stable to heat (100 degrees C for 10 min) and 2-mercaptoethanol reduction but is labile to trypsin digestion. These results emphasize the importance of in vitro test systems to identify bioactive agents which may otherwise go undetected using an in vivo implantation assay.

Partial isolation and characterization of a chemotactic factor from adult bovine bone for mesenchymal cells.

Demineralized adult bone matrix has the capacity to initiate de novo ectopic endochondral bone formation 2-3 weeks following intramuscular implantation into suitable hosts. An early step in this process is the migration of mesenchymal cells to the implant site; these cells later differentiate into cartilage and bone. Adult bone has been shown to contain a number of bioactive factors, such as chemotactic factors for various cell types, including osteoblasts. We have used embryonic chick limb bud mesenchymal cells to construct an in vitro assay for testing chemotactic activity derived from bone matrix extracts. With a modified Boyden chamber, water-soluble components from a 4 M guanidinium chloride extract of demineralized adult bovine bone matrix were found to stimulate the directional migration of these chick embryonic limb bud mesenchymal cells as well as embryonic muscle-derived fibroblasts and cells derived from embryonic skin. The chemotactic activity was destroyed by treatment with heat (100 degrees C) or trypsin. Partial purification by molecular sieve chromatography suggested that the chemotactic factor(s) has a molecular weight of between 50,000 and 90,000. This factor can be separated from bone matrix-derived chondrogenic stimulating activity by either ion exchange or molecular sieve chromatography. These observations confirm that bone matrix contains a chemoattractant for mesenchymal cells that may be important for in vivo recruitment of cells as part of the process of ectopic bone formation or in cases of bone repair.

Isolation and characterization of osteogenic cells derived from first bone of the embryonic tibia.

Osteogenesis in the embryonic long bone rudiment occurs initially within an outer periosteal membrane and subsequently inside the cartilaginous core as a consequence of the endochondral ossification process. In order to investigate the development of these two different mechanisms of bone formation, embryonic chick tibial cell isolates were prepared from sites of first periosteal bone formation and from the immediately underlying hypertrophic cartilaginous core region. Mid-diaphyseal periosteal collars and the corresponding cartilage core were microdissected free from Hamburger-Hamilton stage 35 (Day 9) chick tibias and separately digested with a trypsin-collagenase enzyme mixture. The released cell populations were cultivated in vitro and characterized by morphological analysis, histochemical localization of alkaline phosphatase, alizarin red S staining for mineral deposition, growth rate [( 3H]thymidine uptake), and proteoglycan content. Results of these studies showed that periosteal collar cell cultures form nodule-like structures that stain positive with alkaline phosphatase and alizarin red S. Light and electron microscopic observation revealed cell and matrix morphologies similar to that of intact periosteum. The nodules were composed of plump cell types embedded within a mineralized matrix surrounded by a fibroblastic cell layer. Core cartilage cell cultures displayed typical characteristics of the hypertrophic state in their visual appearance and proteoglycan composition. The formation of osseous-like structures in periosteal collar cell cultures but not in core chondrocyte cell cultures demonstrates the relatively autonomous nature of intramembranous ossification while emphasizing the dependence of the endochondral ossification process upon an intact vascularized environment present in the developing tibia.

The in vitro chondrogenic response of limb-bud mesenchyme to a water-soluble fraction prepared from demineralized bone matrix.

Demineralized adult bone matrix initiates de novo ectopic endochondral ossification 2-3 weeks following its intramuscular implantation into adult animals. This phenomenon appears to be similar, in some ways, to inductive cell-matrix interactions which regulate cartilage and bone formation during development. In the present study, we used embryonic chick limb-bud mesenchymal-cell cultures to bioassay extracts of demineralized bone matrix for chondrogenic activity. Guanidinium-chloride (4 M) extracts of demineralized bovine bone were dialyzed against buffers of decreasing ionic strength and then cold water. The cold-water-soluble fraction was found to stimulate chondrogenesis in intermediate-density limb-bud cell cultures (2.2 X 10(6) cells per 35-mm dish), as revealed by visual inspection with phase optics, toluidine-blue staining of fixed plates, and [35S] sulfate incorporation in the cell layer. Further fractionation of this material by anion-exchange, carbohydrate-affinity, and molecular-sieve chromotography produced a semipurified preparation possessing chondrogenic-stimulating activity at doses ranging from 3 to 10 micrograms/ml. The in vitro chondrogenic response of limb-bud mesenchymal cells was dose-dependent, required a minimal initial plating density of 2.08 X 10(5) cells/mm2 of culture dish, and developed gradually over 8-10 days. At an optimal dose of extract, a continuous exposure period of at least 2-3 days was necessary to produce detectable chondrogenic stimulation. In addition, the amount of cartilage formed following an 8-day exposure was markedly influenced by the culture 'age' of the mesenchymal cells (i.e., the time between plating and the start of treatment).(ABSTRACT TRUNCATED AT 250 WORDS)

An osteo-inductive bone matrix extract stimulates the in vitro conversion of mesenchyme into chondrocytes.

Urea and guanidine extracts of demineralized beef and rabbit bone matrix were assayed both in vivo and in vitro. One month following intramuscular implantation into mouse thighs, these extracts induced ectopic cartilage and bone. Seven days following continuous in vitro exposure to the same extracts, stage 24 chick limb bud mesenchymal cells in cultures had differentiated into greater numbers of chondrocytes than controls. These results suggest the feasibility of using limb bud mesenchymal cell cultures as an in vitro assay for bone matrix derived, extractable bioactive factors which effect the conversion of mesenchymal cells into chondrocytes as a requisite step in in vivo osteogenesis.

A fraction from extracts of demineralized adult bone stimulates the conversion of mesenchymal cells into chondrocytes.

Demineralized adult bone contains factors which stimulate nonskeletal mesenchymal cells to undergo a developmental progression resulting in de novo endochondral ossification. In this study, isolated embryonic stage 24 chick limb bud mesenchymal cells maintained in culture were utilized as an in vitro assay system for detection of specific bioactive components solubilized from adult chicken bone matrix. Guanidinium chloride extracts (4 M) of demineralized-defatted bone were fractionated and tested in limb mesenchymal cell cultures for possible effects upon growth and chondrogenesis. Two low-molecular-weight fractions were found to be active in these cultures. A cold water-insoluble, but warm Tris-buffered saline-soluble fraction provoked a dose-dependent increase in the amount of cartilage formed after 7 days of continuous exposure as evidenced by an increased number of chondrocytes observed in living cultures, elevated cell-layer-associated 35S incorporation per microgram DNA, and greater numbers of toluidine blue-staining foci (i.e., cartilage nodules). Growth inhibitory substances were detected in a low-molecular-weight, water-soluble fraction; 7 days of continuous exposure to this material resulted in less cartilage formation and reduced cell numbers (accumulated DNA) on each plate. These observations demonstrate the usefulness of stage 24 chick limb bud cell cultures for identifying bioactive factors extracted from adult bone matrix. In addition, the action of these factors on mesenchymal cells may now be studied in a cell culture system.

Assessment of an in vivo diffusion chamber method as a quantitative assay for osteogenesis.

The alkaline phosphatase activity and the calcium and phosphorus content of osteogenic tissue formed in vivo following the implantation of diffusion chambers loaded with rabbit bone marrow cells is reported. (In this study the term osteogenic includes osteoblastic and chondroblastic.) Chambers examined 14-70 days after implantation revealed progressive accumulation of mineral. Alkaline phosphatase activity increased until day 30 and declined thereafter. The osteogenic potential of the marrow cells decreased with increasing weight (age) of the cell donor rabbit when measured either as the percentage of chambers containing osteogenic tissue or as the amount of calcium, phosphorus, or alkaline phosphatase activity within the chambers. The results confirm that measurements of these parameters in tissue formed by cells incubated in diffusion chambers in vivo may be used as a method for assay of osteogenesis.

Bone aging.

Bone matrix of 28-month-old (senescent) rats, compared with 13-month-old rats, is strikingly deficient in the capacity to induce bone formation. Similarly, implants of senescent matrix into one-month-old, rapidly-growing rats (weanlings) produce more bone than in 13-month-old adult recipients, but not nearly enough to compensate for deficiency in the matrix of senile bone. Matrix prepared from the bone of 13-month-old rats also produces almost no cartilage and significantly lower yields of new bone in 28-month-old (senescent) than 13-month-old rats. These observations suggest that progressive decline in the matrix morphogenetic activity of bone occurs with time in adult life. This decline is a tissue specific index of bone aging, and may contribute to the reduction of bone mass that normally occurs with senescence.

Effect of hemorrhage on blood flow to marrow and osseous tissue in conscious rabbits.

The effect of hemorrhage on blood flow to marrow and osseous tissue was determined in conscious rabbits by injecting radioactive microspheres (15 micron diam) at three time intervals: pretreatment control; 15 min after nonfatal hemorrhage (20 ml/kg body wt), and 16 h posthemorrhage. Cardiac output (CO), blood pressure, heart rate, resistance, arterial PCO2, PO2, and pH, and hematocrit were measured at each time interval. Tissues analyzed included heart, spleen, kidney, and femur with marrow and osseous tissue separated. At 15 min posthemorrhage, CO decreased to all tissues measured except the heart, where it increased. Sixteen hours later, CO returned to normal, and blood flow increased significantly to heart and marrow, while osseous tissue flow returned to control levels. The results show no immediate shunting of blood from bone to marrow; however, after 16 h there is a selective increase in marrow blood flow. This response is only detected by separating marrow and osseous tissue and it may reflect a preparatory mechanism for increased erythropoietic activity.

Effect of increased oxygen on kidney function in the rat during and after 21 ATA He-O2 exposure.

Two groups of four adult male rats each were exposed continuously for 12 d to 1.2 or 21 ATA He-O2 at 400 mm Hg 02. A third group was maintained under room-air conditions. Urine samples were collected every 12 h for the first 60 h of exposure and for the first 48 h post-decompression. Parameters measured included urine volume, urea, creatinine, calcium, and phosphate. In the 21-ATA group at pressure, urine volume and urea/creatinine increased, and total creatinine decreased; during recovery, total calcium, calcium/creatinine, and urea/creatinine increased and total phosphate decreased. In the 1.2-ATA group, during exposure, urea/creatinine and phosphate/creatinine increased. Diuresis in the 21-ATA group required 48 h to fully develop; however, recovery to normal occurred within 12 h post-decompression. The results reveal a moderate water diuresis and no serious alterations in kidney function during or after 21-ATA exposure at 400 mm Hg O2. There was a mild stress response at pressure and an undefined effect on calcium homeostasis post-decompression.

Skeletal response of growing rats to continuous long-term hyperbaric helium-oxygen exposure.

Bone growth and composition were studied in growing rats following continuous long-term hyperbaric exposure. Six groups of eight rats each were maintained at 21 ATA He-O2 (200 mm Hg O2) and six groups were kept in simulated test chambers under room-air conditions. One group each of pressurized and control animals were removed and analyzed after 2, 3, 5, 8, 10, and 12 weeks. Each animal was weighed and sacrificed. One femur was removed for fresh, dry, ash, and matrix weight measurement and determination of calcium and phosphorus content. The pressurized animals showed a significant reduction in body-weight gain after each exposure period. Femurs from pressurized animals weighed less than controls but had significantly greater femur/body weight percentages. Calcium and phosphorus content was normal and the ratio of matrix to mineral was unchanged. Results suggest that pressurized animals had accelerated metabolic rates and inadequate caloric intake. However, measurements of bone mineral and matrix content indicate the skeleton develops normally under hyperbaric conditions.

Skeletal response of rats to repeated short-term hyperbaric helium-oxygen exposures.

Young male rats were exposed to repeated heliox dives and analyzed for skeletal alterations. Animals were exposed 1, 3, 5, or 7 times to either 1 ATA He-O2 for 12.5 h, or to 5 ATA He-O2 for 4 h and a 8.5 h decompression, or to 5 ATA He-O2 for 4 h and a 1.5 h decompression. In a separate study, 30 rats were exposed 6 times to 5 ATA He-O2 and explosively decompressed. Animals were sacrificed 20 d after the last dive. There were no significant changes in femur wet weight, density, ash weight, length, or mineral content. Plasma calcium, phosphorus, and alkaline phosphatase remained normal. Eighteen of 30 animals survived the six explosive decompressions; however, there were no significant changes in bone. These results indicate that the number and rate of decompressions used in this study have no lasting effect on bone growth and mineral composition in the rat.