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Coarse powders of acid-insoluble matrix of diaphysis and calvarial parietal bone rapidly and consistently transformed fibroblasts into masses of cartilage and bone containing hemopoietic marrow. The transformant was encapsulated by fibroblasts within 24 hr to form a plaque. Transformation was restricted to the central thicknesses of the plaque. Under the stated conditions the alteration of the phenotype, fibroblast to chondroblast, was an unstable transformation, whereas the phenotype change, fibroblast to osteoblast, was stable. The transformation occurred on a rigid timetable of sequences. Measurements of alkaline phosphatase activity and incorporation of radioactive sulfate, phosphate, and calcium were sensitive and quantitative assays for the appearance of the transformed products, cartilage and bone.The aim of this work was to develop reproducible, rapid, and quantitative methods (a) to induce the fibroblast-chondroblast-osteoblast transformation, and (b) to differentiate the major links in the biological sequence.Long after embryonic differentiation has ceased, fibroblasts still retain the singular potential of transformability (1) into cells of other sorts, an attribute that persists throughout the life of the animal. The visible and biochemical characters are altered so profoundly that we refer to the phenomenon as transformation (2). Approximation of transformant (TF) and competent responding fibroblasts (R) initiates a series of interconnected biological reactions that yield products which we shall designate: Pi, cartilage; P2, bone; P3, hemopoietic bone marrow.Urist discovered that intramuscular transplants of lyophilized segments of demineralized bone (3) or tooth (4) transform fibroblasts to form bone by endochondral ossification in 24-26 days. Chondrogenesis occurs in cell culture (5), as well as in vivo. There are two convenient enzyme assays to study the transformation of the fibroblasts of fascia into cartilage, followed by bone: a. activity of alkaline phosphatase (6); b. determination of the quotient of activity: lactate dehydrogenase/malate dehydrogenase (7).The present experiments consisted of allogeneic transplantation of a weighed amount of sized desiccated powder of acidinsoluble bone matrix to the subcutaneous tissues of young rats. This technique provided a simple, quick, and standardized method to induce the transformation. The biochemical sequences of cartilage and bone in the chain reaction were analyzed by measurement of enzyme activities and incorporation of isotopes in the transformation products. MATERIALS AND METHODSPreparation of Transformant. Manufacture and final storage of all preparations were at room temperature (about 250).When liquids of any sort were used, the biological materials were immersed in the fluids in a jar with a magnetic stirrer, where they were propelled around the vortex created by vigorous stirring.Large adult rats of both sexes were used as donors throughout. The rats were decapitated. Parietal bones of the calvarium were removed and fragmented. ...

Morphogenesis is the developmental cascade of pattern formation, body plan establishment, and the architecture of mirror-image bilateral symmetry of many structures and asymmetry of some, culminating in the adult form. Tissue engineering is the emerging discipline of design and construction of spare parts for the human body to restore function based on principles of molecular developmental biology and morphogenesis governed by bioengineering. The three key ingredients for both morphogenesis and tissue engineering are inductive signals, responding stem cells, and the extracellular matrix. Among the many tissues in the human body, bone has considerable powers for regeneration and is a prototype model for tissue engineering based on morphogenesis. Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. This model mimics sequential limb morphogenesis and permitted the isolation of bone morphogens. Although it is traditional to study morphogenetic signals in embryos, bone morphogenetic proteins (BMPs), the inductive signals for bone, were isolated from demineralized bone matrix from adults. BMPs and related cartilage-derived morphogenetic proteins (CDMPs) initiate, promote, and maintain chondrogenesis and osteogenesis and have actions beyond bone. The symbiosis of bone inductive and conductive strategies are critical for tissue engineering, and is in turn governed by the context and biomechanics. The context is the microenvironment, consisting of extracellular matrix, which can be duplicated by biomimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhesion proteins including fibronectins. Thus, the rules of architecture for tissue engineering are an imitation of the laws of developmental biology and morphogenesis, and thus may be universal for all tissues, including bones and joints.

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Prostate cancer is the second most common cause of death from cancer in U.S. men, and advanced, hormone-refractory disease is characterized by painful osteoblastic bone metastases. Endothelin-1, more commonly known as a potent vasoconstrictor, is a normal ejaculate protein that also stimulates osteoblasts. We show here that plasma immunoreactive endothelin concentrations are significantly elevated in men with metastatic prostate cancer and that every human prostate cancer cell line tested produces endothelin-1 messenger RNA and secretes immunoreactive endothelin. Exogenous endothelin-1 is a prostate cancer mitogen in vitro and increases alkaline phosphatase activity in new bone formation, indicating that ectopic endothelin-1 may be a mediator of the osteoblastic response of bone to metastatic prostate cancer.

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...

Human postnatal bone marrow stromal stem cells (BMSSCs) have a limited life-span and progressively lose their stem cell properties during ex vivo expansion. Here we report that ectopic expression of human telomerase reverse transcriptase (hTERT) in BMSSCs extended their life-span and maintained their osteogenic potential. In xenogenic transplants, hTERT-expressing BMSSCs (BMSSC-Ts) generated more bone tissue, with a mineralized lamellar bone structure and associated marrow, than did control BMSSCs. The enhanced bone-forming ability of BMSSC-Ts was correlated with a higher and sustained expression of the early pre-osteogenic stem cell marker STRO-1, indicating that telomerase expression helped to maintain the osteogenic stem cell pool during ex vivo expansion. These results show that telomerase expression can overcome critical technical barriers to the ex vivo expansion of BMSSCs, and suggest that telomerase therapy may be a useful strategy for bone regeneration and repair.

Progress in understanding the role of bone morphogenetic proteins (BMPs) in craniofacial and tooth development, the demonstration of stem cells in dental pulp and accumulating knowledge on biomaterial scaffolds have set the stage for tissue engineering and regenerative therapy of the craniofacial complex. Furthermore, the recent approval by the US Food and Drug Administration (FDA; Rockville, MD, USA) of recombinant human BMPs for accelerating bone fusion in slow-healing fractures indicates that this protein family may prove useful in designing regenerative treatments in dental applications. In the near term, these advances are likely to be applied to endodontics and periodontal surgery; ultimately, they may facilitate approaches to regenerating whole teeth for use in tooth replacement.