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Fixation and demineralization protocols for bone marrow (BM) across diagnostic laboratories are not standardized. How different protocols affect histomorphology and DNA amplification is incompletely understood. In this study, 2 fixatives and 3 demineralization methods were tested on canine BM samples. Twenty replicate sternal samples obtained within 24 hours of death were fixed overnight in either acetic acid-zinc-formalin (AZF) or 10% neutral-buffered formalin (NBF) and demineralized with formic acid for 12 hours. Another 53 samples were fixed in AZF and demineralized with hydrochloric acid for 1-hour, formic acid for 12 hours, or ethylenediamine tetraacetic acid (EDTA) for 24 hours. Histologic sections were scored by 4 raters as of insufficient, marginal, good, or excellent quality. In addition, DNA samples extracted from sections treated with the different fixation and demineralization methods were amplified with 3 sets of primers to conserved regions of T cell receptor gamma and immunoglobulin heavy chain genes. Amplification efficiency was graded based on review of capillary electrophoretograms. There was no significant difference in the histomorphology scores of sections fixed in AZF or NBF. However, EDTA-based demineralization yielded higher histomorphology scores than demineralization with hydrochloric or formic acid, whereas formic acid resulted in higher scores than hydrochloric acid. Demineralization with EDTA yielded DNA amplification in 29 of 36 (81%) samples, whereas demineralization with either acid yielded amplification in only 2 of 72 (3%) samples. Although slightly more time-consuming and labor-intensive, tissue demineralization with EDTA results in superior morphology and is critical for polymerase chain reaction (PCR) amplification with the DNA extraction method described in this article., Competing Interests: Declaration of Conflicting InterestsThe author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Clinical application of a demineralized freeze-dried cortical bone membrane allograft (DFBMA) for treatment of intra(infra)bony periodontal pockets in dogs was evaluated. The mean pre-treatment periodontal probing depth equaled 7.2-mm. Post-treatment probing depths in all 11 cases were normal, with a mean periodontal probing gain of 5.4-mm. Guided tissue regeneration using a commercially available veterinary canine DFBMA and canine demineralized freeze-dried bone allograft (DFDBA) resulted in clinically significant periodontal attachment gains. The gain of new periodontal tissue attachment was statistically significant (P < 0.0001). The commercially available veterinary allograft products predictably increased new periodontal attachment without any identified membrane sequelae in these 11 cases.

Canine mesenchymal cells (MSCs) derived from Wharton's jelly were co-cultured, then supplemented or not supplemented with platelet rich plasma (PRP) and demineralized bone matrix (DBM) to verify osteogenic differentiation. Osteoblastic differentiation followed by mineralized bone matrix production was found to be significantly higher (p < 0.05) when MSCs were associated with PRP/DBM in culture after 14-21-days of induction. Osteopontin and osteocalcin gene expression were significantly superior (p < 0.05) under the same culture conditions after 21 days of observation. In conclusion, addition of PRP to DBM co-cultured with MSCs successfully induced osteogenesis in vitro.

Objective: To evaluate the osteogenic potential of avian demineralized bone matrix (DBM) in the context of implant geometry., Study Design: Experimental., Animals: Rock pigeons (n = 24)., Methods: Tubular and chipped forms of DBM were prepared by acid demineralization of long bones from healthy allogeneic donors and implanted bilaterally into the pectoral region of 24 pigeons. After euthanasia at 1, 4, 6, 8, 10, and 12 weeks, explants were evaluated histologically and compared by means of quantitative (bone area) and semi quantitative measures (scores)., Results: All explants had new bone at retrieval with the exception of tubular implants at the end of week 1. The most reactive part in both implants was the interior region between the periosteal and endosteal surfaces followed by the area at the implant-muscle interface. Quantitative measurements demonstrated a significantly (P = .012) greater percentage of new bone formation induced by tubular implants (80.28 ± 8.94) compared with chip implants (57.64 ± 3.12). There was minimal inflammation., Conclusions: Avian DBM initiates heterotopic bone formation in allogeneic recipients with low grades of immunogenicity. Implant geometry affects this phenomenon as osteoconduction appeared to augment the magnitude of the effects in larger tubular implants., (© Copyright 2013 by The American College of Veterinary Surgeons.)

The osteoinductive capability of four particle sizes of equine demineralized bone matrix (DBM) was evaluated. Matrix particles were implanted in brachiocephalicus muscle pouches in six horses and were harvested 8 weeks later. Matrix particle sizes of 2.0 mm3 to 4.0 mm3 and 5.0 mm3 to 10.0 mm3 were associated with osteoinductive activity and minimal signs of local inflammation. The two smaller particle sizes (0.425 mm3 to 0.850 mm3 and 0.850 mm3 to 2.0 mm3) were minimally osteoinductive and were associated with a greater local inflammatory response. Microscopic events associated with new bone production in the two largest particle sizes included the presence of new osteoprogenitor cells in matrix vascular spaces, multinucleated osteoclast-like giant cells, and the formation of cartilage, osteoid, and new mineralized bone. The two smaller particle sizes were associated with increased fibrous tissue ingrowth and the presence of increased numbers of inflammatory cells. The temporal events of osteoinduction in response to heterotopic implantation of allogeneic DBM were delayed in horses when compared with other species. Although the demineralization technique used was effective in previous investigations on other species, incomplete demineralization occurred in the two smaller particle sizes that was not detected until after DBM implant harvest. The demineralization process may need to be altered for equine bone and precise monitoring for complete demineralization before implantation is required. Equine DBM is osteoinductive in muscle pouch sites in horses but matrix particle size does affect osteoinductive capability.