Quantification of various growth factors in different demineralized bone matrix preparations

Abstract

Besides autografts, allografts, and synthetic materials, demineralized bone matrix (DBM) is used for bone defect filling and treatment of non-unions. Different DBM formulations are introduced in clinic since years. However, little is known about the presents and quantities of growth factors in DBM. Aim of the present study was the quantification of eight growth factors important for bone healing in three different "off the shelf" DBM formulations, which are already in human use: DBX putty, Grafton DBM putty, and AlloMatrix putty. All three DBM formulations are produced from human donor tissue but they differ in the substitutes added. From each of the three products 10 different lots were analyzed. Protein was extracted from the samples with Guanidine HCL/EDTA method and human ELISA kits were used for growth factor quantification. Differences between the three different products were seen in total protein contend and the absolute growth factor values but also a large variability between the different lots was found. The order of the growth factors, however, is almost comparable between the materials. In the three investigated materials FGF basic and BMP-4 were not detectable in any analyzed sample. BMP-2 revealed the highest concentration extractable from the samples with approximately 3.6 microg/g tissue without a significant difference between the three DBM formulations. In DBX putty significantly more TGF-beta1 and FGFa were measurable compared to the two other DBMs. IGF-I revealed the significantly highest value in the AlloMatrix and PDGF in Grafton. No differences were accessed for VEGF. Due to the differences in the growth factor concentration between the individual samples, independently from the product formulation, further analyzes are required to optimize the clinical outcome of the used demineralized bone matrix.

Treatment of osteochondral defects of the talus

“…85,234,399 Th ese joints might thus not be reliably compared in osteochondral defect healing models. In general, the eff ectiveness of DBM depends on numerous variables, including the age of the donor, 347 the tissue bank and the lot, 172,173,450 prolonged heat treatment, 196 the size or shape of the graft particles, 155 the nature of the carrier material, the extent of residual bone mineral content, 459 and the method of sterilization. 171 However, most of these variables are not applicable to the commercially available DBM used in the current study.…”

Wnt pathway regulation by demineralized bone is approximated by both BMP‐2 and TGF‐β1 signaling

“…Although BMP-2 was originally isolated as a putative inductive factor in DBP, these results show that rhBMP-2 and DBP do not affect all the Wnt components in the same ways. Because Guanidine HCl/ EDTA extracts of demineralized bone contain BMP-2, BMP-4, BMP-7/OP-1, TGF-b1, IGF-I, and other biologically active proteins, 21,22 we proposed that it was unlikely that a single component could duplicate all of the effects of DBP. 6,10 We reported previously that DBP and BMP-2 regulate common and distinct signaling pathways, that DBP stimulates both Smad pathways, the one responsive to TGF/Activin and the one responsive to BMP, 10 and that DBP evoked early changes in Wnt components.…”

“…Concentrations of cDNA and amplification conditions were optimized for each gene product to reflect the exponential phase of amplification. One microliter of the cDNA solution was used in each PCR (21)(22)(23)(24)(25)(26)(27)(28)(29)(30)(31)(32)(33)(34)(35) cycles at 948C for 1 min, 55 or 608C for 1 min, and 728C for 2 min) with human gene specific primers: bigh3/TGFBI and ID3, 10 WNTs, 11 Cbfa1/Runx2 and Col1A1, 18 and SOX9. 19 …”

An overview on bone protein extract as the new generation of demineralized bone matrix

“…Carrington et al [38] demonstrated that TGF-βs were present in DBM following extraction with EDTA-guanidine hydrochloride, but not guanidine hydrochloride alone. Wildemann et al [35] verified that TGF-β1 was present in all the three commercial DBMs, although it was at a low level. Katz et al [18] found a weak Spearman correlation between the level of TGF-β1 in the DBM of donors' bone and its capacity for bone formation.…”

“…Wildemann et al [35] demonstrated that DBM, which has been demineralized and sterilized by different processing methods, retained different amounts of insoluble proteins by comparing three commercially available DBMs. However, in those studies, BMP-2 was detected at its highest level in all DBMs tested.…”