Dissociative extraction and reconstitution of extracellular matrix components involved in local bone differentiation.

Abstract

Subcutaneous implantation ofdemineralized diaphyseal bone matrix in allogeneic rats results in the local induction of endochondral bone differentiation. We have explored the potential of three dissociative extractants, 4 M guanidine hydrochloride (Gdn HCI), 8 M urea/i M NaCI, and 1% NaDodSO4 at pH 7.4, containing protease inhibitors to solubilize putative inductive molecules in the bone matrix. Extraction of bone matrix with any one of these extractants resulted in the loss of the bone inductive property. The solubilized extracts were then reconstituted with the residue by dialysis against water. The various reconstituted matrices were bioassayed for bone inductive potential by quantitation of alkaline phosphatase activity and 4Ca incorporation on day 12 after implantation. There was complete recovery of biological activity after reconstitution of the residues with each of the three extracts. Polyacrylamide gel electrophoresis of the extracts revealed similar protein profiles. Gel filtration of the 4 M GdnwHCl extract on Sepharose CL-4B showed a heterogeneous broad peak. When fractions of that peak containing proteins <50,000 daltons were reconstituted with inactive 4 M Gdn HCItreated bone matrix and then implanted, new bone was induced. These observations demonstrate the dissociative extraction and successful biological reconstitution of bone inductive macromolecules in demineralized bone matrix.Subcutaneous implantation of demineralized diaphyseal bone matrix in allogeneic rats results in the local differentiation of fibroblasts to form endochondral bone (1-3). The developmental cascade of endochondral bone differentiation consists of mesenchymal cell chemotaxis, cell proliferation, differentiation and hypertrophy of chondrocytes, vascular invasion, calcification ofcartilage matrix, formation and remodeling ofbone, and, finally, differentiation of hematopoietic bone marrow in the newly formed ossicle (4). Although the cellular and biochemical events accompanying the sequential cell differentiation are well known (4), the precise molecular mechanisms underlying the action ofmatrix on cells are not clear. The present report describes the dissociative extraction and successful biological reconstitution of extracellular matrix components involved in induction of bone differentiation. MATERALTS AND METHODSPreparation of Demineralized Bone. Dehydrated diaphyseal shafts of rat femur and tibia were pulverized in a CRC micromill (Techni Laboratories, Vineland, NJ) and sieved to a discrete particle size of 74-420 Aum. The powders were demineralized with 0.5 M HCl, extracted with water, ethanol, and ether, and prepared as described (1). (20 mM Na2HPO450 mM Tris, pH 7.4), essentially the same extractant but with 0.25% Triton X-100, and three different dissociative extractants: 4 M Gdn.HCV50 mM Tris, pH 7.4; 8 M urea/i M NaCV50 mM Tris, pH 7.4; 1% NaDodSO4/50 mM Tris, pH 7.4. All extractants contained a mixture of protease inhibitors: 5 mM benzamidine/0.1 M 6-aminohexanoic acid/ 0.5 mM phenylmethylsulfonyl fluorid...

Repair of Ulnar Segmental Defect by Recombinant Human Bone Morphogenetic Protein-2 in Dogs.

“…Since osteoinduction by demineralized bone matrix (DBM) was first reported by Urist in 1965, the potency of DBM or extracted BMPs as a bone graft substitute have been well investigated in rodents [17,21,24]. However, the effect of DBM in non-rodent mammals is uncertain, with failure of osteoinduction using DBM having been reported in both dogs [18] and monkeys [1].…”

“…The transient clinical symptoms observed during the first 2 weeks after surgery were apparently elicited by the implanted rhBMP-2. This period corresponds to the phase from mesenchymal cell proliferation to subsequent differentiation into osteogenic cells [17,25,28]. These marked responses of host tissues might be considered as clinical signs resembling inflammatory responses.…”

“…BMP was first postulated by Urist in 1965 [23], and was subsequently extracted chemically from demineralized bone matrix [17,24]. Wozney et al [27] employed recombinant DNA technology to produce the proteins in 1988.…”

The Otto Aufranc Award: Demineralized Bone Matrix Around Porous Implants Promotes Rapid Gap Healing and Bone Ingrowth

“…We combined both matrices with 100% glycerol as a carrier in a 1-to-4 ratio, producing graft materials with a putty-like consistency. By comparing total protein content using established protocols, we were able to compare growth factor content between the DBM and its devitalized form [2,3,30]. Protein extraction solution consisted of guanidine HCl 4 mol/L, Tris 50 mmol/L, and a protease inhibitor cocktail with EDTA 1 mmol/L (SigmaAldrich, Oakville, Canada).…”

“…DBM, produced by acid extraction of the mineralized phase of bone to create a composite of noncollagenous proteins, growth factors, and collagen [12], is capable of inducing osteogenesis by recruiting host mesenchymal stem cells [37]. Various commercial preparations of DBM are available, differing widely in their ability to induce bone formation due to different processing techniques and carriers [10,11,28,30,31,36]. Evaluation of the potential for DBM to enhance bone ingrowth or promote gap healing around porous-coated implants is limited.…”