Dystrophic calcification and mineralization during bone induction: biochemical differences

The calcification of implants of glutaraldehyde-crosslinked collagenous tissues and collagen was studied in young and old rats and compared to bone induction by non-crosslinked osteogenically active demineralized bone matrix (DBM). Glutaraldehyde-crosslinked implants of DBM, tendon, and cartilage calcified in young but not in old animals and accumulated only trace amounts of BGP (Bone Gla protein, osteocalcin). Alkaline phosphatase activity was high in implants of DBM and undetectable in crosslinked implants. To try and understand why bone formation is so significantly reduced in older Fischer-344 rats, we developed a system which consists of cylinders of DBM sealed at the ends with a Millipore filter. Cells originating from 20-day-old embryo donors were introduced into the chambers prior to subcutaneous implantation. After 4 weeks of implantation in 26-month-old rats, the cylinders containing embryonic calvaria or muscle calls were found to be full of bone and/or cartilage.     

Enzyme patterns during bone induction

Results and conclusions The results are as yet preliminary. The tendency for quantitative variation is given in Fig. 1 and the relative variation in isoenzyme pattern of LDH in Fig. 2. LDH activity increased parallel with increased level of metabolism and had a marked activity located to osteoblasts when new bone formation started. The isoenzyme pattern changed from an anaerobic one at day 14 towards an aerobic one as capillary ingrowth occurred. Interestingly enough we, like earlier workers, have found a considerable. AklPase activity in the very early stages (14 and 18 days) long befor any sign of new bone formation was evident. This activity is always in strict contact with the old matrix, in a zone approximatelly 200–500 micra populated by osteoprogenitor cells. AcPase activity shows one peak in the early sequence when it can be located to multinuclear macrophages (also pos. for succinic dehydrogenase) and a second peak almost simultaneously with the formation of new bone reflecting the remodelling phase. Activity is then mainly in osteoclasts with some also found in osteoblasts. Further findings will be brought forward in the discussion.     

Bone matrix-induced local bone induction

The sequential cellular changes in the implants in response to collagenous bone matrix-induced local bone formation include: binding of fibronectin to matrix, chemotaxis and attachment of progenitor cells, proliferation and differentiation of progenitor cells into chondrocytes, and finally osteogenesis and marrow differentiation. The cellular origin of osteogenic proteins is not clear. The present study compares the osteogenic potential of demineralized rat and porcine bone matrix by dissociative extraction and reconstitution. Judging from the Sephacryl S-200 gel filtration profiles of the dissociative extracts of rat and porcine matrix, the latter appears to be smaller. Under identical experimental conditions, the rat chondrosarcoma and osteosarcoma were examined for chondrogenic and osteogenic properties and found to be devoid of inductive potential. It is noteworthy that gel filtration fractions of rat chondrosarcoma on Sepharose CL-6B are inhibitory to bone inductive potential of demineralized rat bone matrix.     

Experimental induction of heterotopic bone

Heterotopic bone can be induced in experimental animals by trauma to the soft tissues, by induction from living cells, or by extracts from bone and teeth. In the first two types, the mechanism of the inductive process is not known, whereas in the latter, a factor isolated from bone matrix induces bone formation. Mesenchymal cells in bone marrow are determined for development into cartilage and bone cells and only an unspecific stimulus, such as trauma or autotransplantation, is sufficient for the development into mature osteogenic tissue. Mesenchymal cells will not differentiate into bone cells unless stimulated by a specific inductive substance, bone morphogenetic protein (BMP). Implanted to a heterotopic site, BMP induces undifferentiated mesenchymal cells into a bone morphogenetic pathway of development, causing heterotopic bone formation. The quantitative inductive response is dependent on the source of the BMP, and the bone formation is also determined by the recruitment of inducible target cells and by the environment at the implantation site. Hence, the environment at the implantation site is of major importance for the amount of bone formed. BMP initiates a cascade of events that is modulated by endocrine and paracrine factors. The heterotopic bone has all the morphologic and biochemical characteristics of orthotopic bone, is subjected to turnover, and even has the intriguing ability to generate the formation of bone marrow. Experimental induction of heterotopic bone has become a most useful method to study osteoneogenesis and has supplied important information on the prerequisites for new bone formation and on the regulation of bone metabolism.     

Biologic principles of bone induction

This article provides a concise review of bone induction. Bone induction by demineralized bone matrix is a multistep cascade. The purification and elucidation of the chemistry of osteogens will improve bone grafting methods.     

Age effects on bone induction by demineralized bone powder

It has been previously shown that osteoinduction by demineralized bone powder (DBP) in the rat decreases as the age of the recipient animal increases. In the present study, the effects of age on osteoinduction by DBP were evaluated in the rat by varying the age of the donor and the recipient animal. Cartilage and bone formation in subcutaneous pouches was assessed using a histologic grading technique in which a composite score was derived from analysis of multiple histologic sections from each specimen. The results confirm the previously reported decrease in osteoinduction in middle-aged adult animals compared with younger ones. However, DBP prepared from middle-aged adult rats was more inductive than that prepared from either prepubertal or young postpubertal animals. The latter result contradicts the widely held belief that demineralized bone matrix from younger animals is more inductive than that from older ones. This finding may help to further elucidate the mechanism of ectopic bone formation and lead to more inductive bone graft substitutes for human use.     

Bone induction with bone morphogenic protein

32 experiments in 22 rabbits were performed to investigate the bone regeneration after implantation of lone morphogenetic protein (BMP) in a 6 mm drill-hole of the distal femoral condyle. Two different BMP products have been used. A bone regeneration is to be observed partially at the surface of BMP, but in comparison to the control defects quantitatively insignificant. There is a significant difference in comparison to the bone substitutes Collapat and Pyrost, which proved well (highly significant more bone regeneration) in the same experimental set up. The biology of bone regeneration by; 1. osteogenic transplantation; 2. osteoinduction and 3. osteoconduction ist discussed in detail. According to the own experiments the principle of osteoinduction with BMP is not yet useful for general clinical practice. Probably the osteoinductive effect is disturbed by immunological processes. The extraction of pure "osteogenin" from BMP seems to be important.     

Failure of bone induction by bone matrix in adult monkeys

Extraskeletal bone formation can be induced in rodents by implantation of demineralised bone matrix and such implantation has been used to treat bone defects in man, but it is uncertain if induction or merely conduction occurs. We studied bone induction in primates by excising segments of the fibulae of adult squirrel monkeys, defatting and demineralising them before reimplanting them into the quadriceps of the same animal. As a control experiment, rat matrix was prepared in exactly the same way and implanted in rats. After six weeks the implants were harvested and either ashed and analysed for calcium content or prepared for histology. In the rats, the calcium content indicated that about 20% of the original matrix had been replaced by new bone. In the monkeys the calcium content was about the same as that in normal body fluid and no bone was seen in histological sections. This result casts doubt on the use of demineralised human bone matrix as a bone inductor, although it may function by other mechanisms.     

A comparison: The efficacy of sevoflurane-nitrous oxide or propofol-nitrous oxide for the induction and maintenance of general anesthesia

Study Objective: To compare sevoflurane-nitrous oxide with propofol-nitrous oxide for the induction and maintenance of anesthesia, and to determine the rates of recovery following each anesthetic.Design: Randomized, controlled study.Setting: Teaching hospital.Patients: 50 ASA physical status I and II patients, ranging in age from 18 to 70 years.Interventions: General anesthesia was induced with either sevoflurane or propofol and maintained with 60 % to 70% nitrous oxide and either sevoflurane or a propofol infusion and supplemental fentanyl. At the conclusion of surgery, the oxygen flow was increased to 6 L/min and all anesthetics were discontinued simultaneously. Patients were monitored for the nature and speed of induction and emergency from anesthesia.Measurements and Main Results: Induction of anesthesia was significantly slower in the sevoflurane group than in the propofol group (2.0 ± 1.1 vs. 0.8 ± 0.5 min, respectively). The ease of induction and the time required for emergence from anesthesia were the same in both study groups (eye opening: 9.0 ± 4.4 min vs. 8.0 ± 5.0 min; following commands: 11.2 ± 5.0 min vs. 9.8 ± 6.9 min; extubation: 9.1 ± 4.5 min vs. 8.6 vs. 5.1 min in the sevoflurane and propofol groups, respectively). Patients in the sevoflurane group experienced nausea and vomiting more frequently than patients in the propofol group (13 and 5 patients vs. 3 and 0 patients in the sevoflurane and propofol groups, respectively), which were not related to the administration of neostigmine or intraoperative opioids.Conclusion: Sevoflurane allows for rapid inhalation induction of, and emergence from, general anesthesia.     

Subchondral bone sclerosis and cancellous bone loss following OA induction depend on the underlying bone phenotype

Objectives

Osteoarthritis (OA) is increasingly considered a disease of the whole joint, yet the interplay between the articular cartilage and the subchondral bone remains obscure. We here set out to investigate the impact of bone mass on the progression of surgically induced knee OA in the mouse.

Basic fibroblast growth factor and bone induction in rats

Bone induction is initiated by bone morphogeneic proteins, but local growth factors present in deminer-alized bone matrix (DBM) may further regulate the process. We have previously shown that local application of recombinant human basic fibroblast growth factor (bFGF) in a carboxymethyl cellulose gel to DBM implants increases the bone yield, as measured by calcium content. In the present study, similar experiments were evaluated with histomorphometry. The chondrocyte number at 2 weeks was increased by the application of 15 ng bFGF. This increase was due to an increased number of chondrocyte clusters, i.e., cartilage formation was initiated in more places within the implant. The size of the individual chondrocyte clusters was the same as in the controls. Thus, the bFGF had probably stimulated cellular events preceding chondrocyte proliferation. At 3 weeks, the chondrocytes were fewer than in controls, and instead there was more bone. Thus, cartilage formation was increased by bFGF, and its replacement by bone came earlier. However, 1900 ng of bFGF had a profound inhibitory effect on both cartilage and bone formation.     

Cellular events associated with the induction of bone by demineralized bone

Implantation of demineralized bone (DB) in the form of powder or intact segments in extra skeletal sites stimulates new bone formation. Urist and co-workers presented substantial evidence that there is a noncollagenous protein that has the ability to induce bone formation. One aim of this study was to trace the process of bone formation when DB, in the form of perforated rectangular plates, is implanted subcutaneously in 2-month-old rats. A second objective was to determine whether cartilage cells play a role in the formation of bone in this model. Various DB plates with 0.25 mm diameter holes were implanted subcutaneously for 1-4 weeks in rats. One week after implantation, DB plates were covered by vascularized connective tissue that invaded the perforations. Aggregates of chondrocytes were observed within the holes and on periosteal surfaces in only a few specimens. Further cartilage proliferation was not observed, and by the 2nd week there was no evidence of endochondral bone formation. Where these cartilage-like cells were present, a thin layer of mineral was deposited around them; resorption and fibrous tissue infiltration followed. This aborted form of endochondral calcification was not followed spatially by bone formation. Patent vascularized channels were invaded by alkaline phosphatase-positive mononuclear cells and fibroblasts, and became enlarged by the enzymatic action of macrophages. The next step involved the calcification of DB plates adjacent to the wide spaces. Osteoclasts now appeared leading to the resorption of this recalcified matrix. The eroded and now enlarged lacunar surfaces were lined by newly formed bone and osteoblasts. This process continued so that, at the end of 4 weeks following implantation, the original DB plates were replaced by trabecular bone. Biochemical data on calcium and alkaline phosphatase levels in the implants paralleled the morphological observations.