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Morphogenesis and Tissue Engineering of Bone and Cartilage: Inductive Signals, Stem Cells, and Biomimetic Biomaterials
A.H. REDDI, Ph.D.
ABSTRACT 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. INTRODUCTION
is to convey recent progress in the morphogenesis of bone and its induction by bone morphogenetic proteins (BMPs) and applications in tissue engineering. The logic of this approach is based on the premise that understanding of molecular principles of development and morphogene sis of a tissue will provide the rationale for enunciating the rules of architecture for tissue engineering. We deHE AIM O F TH IS AR TIC LE
Center for Tissue Regeneration and Repair and Department of Orthopaedic Surgery, University of California, Davis, Medical Center, Sacramento, California.
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REDDI fine tissue engineering as the science of design and manufacture of new tissues for the functional restoration of the impaired organs and replacement of lost parts due to disease, trauma, or tumors. Tissue engineering is based on principles of cellular and molecular developmental biology and morphogenesis guided by bioengineering and biomechanics. The three key ingredients for both morphogenesis and tissue engineering are inductive signals, responding stem cells, and the extracellular matrix. The extracellular matrix is the context (microenvironm ent) and can be functionally duplicated by a supramolecular assembly (scaffolding) of biomimetic biomaterials such as collagens, proteoglycans, and cell adhesion glycoprotein s such as fibronectins and laminin. One of the consequences of the aging process in humans is the inevitable impaired locomotion due to bone and joint problem s due to osteoporosis and arthritis. Among the many tissues in the body, bone has considerable potential for repair and, therefore, is a natural paradigm for delving into the mysteries of regeneration and morphogenesis. Morphogens are generally first identified in fly and frog embryos by genetic approaches, differential displays, substractive hybridization , and expression cloning, and this inform ation is then extended to mice and humans. This article will demonstrate an alternative biochemical approach based on regenerative potential of adult mammalian bone. The principles gleaned from bone induction and BMPs have been extended to frog mesoderm induction and chick limb morphogenesis. This accrued knowledge can now be applied to tissue engineering creation of spare parts for the human body based on inductive signals, responding stem cells, and biomimetic biomaterials based on extracellular matrix.
MORPHOGENESIS AND TISSUE ENGINEERING A major stumbling-block in the approach was that the demineralized bone matrix is insoluble. In view of this, dissociative extractants such as 4 M guanidine HCl or 8 M urea as 1% sodium dodecyl sulfate (SDS) at pH 7.4 were used. 9 Approximately 3% of the proteins were solubilized from demineralized bone matrix, and the remaining residue was mainly insoluble type I bone collagen. The soluble extract alone or the insoluble residue alone was incapable of new bone induction. However, addition of the extract to the residue (insoluble collagen), and then implantation in a subcutaneous site resulted in bone induction. Thus, it would appear that for optimal osteogenic activity there was a collaboration between soluble extract and the insoluble collagenous substratum .9 This bioassay was a useful advance in the final purification of BMPs and led to determination of limited tryptic peptide sequences leading to the eventual cloning of BMPs. 10 12
REDDI nidus for replacement by bone, and are the organizer centers of longitudinal and circumferental growth of cartilage setting into motion the orderly developm ental program of endochondra l bone formation. 5 The phenotypic stability of the articular (permanent) cartilage is at the crux of the osteoarthritis problem. The maintenance factors for articular chondrocytes include TGF- b isoforms and the BMP isoforms. An in vivo chondrogenic bioassay with soluble purified proteins and insoluble collagen identified a chondrogenic fraction in articular cartilage.16 A concurrent reverse transcriptase polymerase chain reaction (RTPCR) approach with degenerate oligonucleot ide primers was undertaken. Two novel genes for cartilage-derived morphogenetic proteins (CDMPs) 1 and 2 were identified and cloned. 16 CDMPs 1 and 2 are also called GDF-5 and GDF-617 and may play a critical role in initiation and maintenance of articular cartilage and joint morphogenesis.16,17
MORPHOGENESIS AND TISSUE ENGINEERING ical role of this molecule in kidney and eye developm ent.26,27 Thus, the BMPs are really true morphogens for such disparate tissues as skin, heart, kidney, and eye.
BIOMIMETIC BIOMATERIALS
The earlier discussion of inductive signals (BMPs) and responding stem cells (stromal cells) leads us to the scaffolding (the microenvironm ent/extracellular matrix) for optimal tissue engineering. The natural biomaterials in the composite tissue of bones and joints are collagens, proteoglyca ns, and glycoprotein s of cell adhesion such as fibronectin and the mineral phase. The mineral phase in bone is predominantly hydroxyapatite, a common geomineral of calcium phosphate. The high protein binding capacity makes hydroxy355
REDDI apatite a natural delivery system. Comparison of insoluble collagen, hydroxyapati te, tricalcium phosphate, glass beads, and polym ethylmethacrylate as carriers revealed collagen to be an optim al delivery system for BMPs. 40 It is well known that collagen is an ideal delivery system for growth factors in soft and hard tissue wound repair. 41 During the course of systematic work on hydroxyapatit e of two pore sizes (200 or 500 m m) in two geometrical forms (beads or discs), an unexpected observation was made. The geometry of the delivery system is critical for optim al bone induction. The discs were consistently osteoinductive with BMPs in rats, but the beads were inactive. 42 The chemical compositions of the two hydroxyapatit e configurations were identical. In certain species, the hydroxyapatit e alone appears to be osteoinductive. 43 In subhuman primates, the hydroxyapatit e induces bone albeit at a much slower rate. One interpretation is that osteoinductive endogenous BMPs in circulation progressively bind to the implanted disc of hydroxyapati te. When an optimal threshold concentration of native BMPs is achieved, the hydroxyapat ite becomes osteoinductiv e. Strictly speaking, most hydroxyapati te substrata are ideal osteoconduc tive materials. This example in certain species also serves to illustrate how an osteoconduc tive biomimetic biomaterial may progressively function as an osteoinductiv e substance by binding to endogenous BMPs. Thus, there is a physiologica l-physicochemical continuum between the hydroxyapatit e alone and progressive composites with endogenous BMPs. Recognition of this experimental nuance will save unnecessary arguments amongst biomaterials scientists about the osteoinductive action of a conductive substratum such as hydroxyapati te. Complete regeneration of a baboon craniotomy defect was accomplished by recombinant human osteogenic protein (rhOP-1; human BMP-7). 44 Recombinant BMP-2 was delivered by a poly( a -hydroxy acid) carrier for calvarial regeneration. 45 Copolym ers of polylactic and plyglycolic acid, or carrier for BMP-2 with excellent bone formation in a nonunion model in rabbit ulna, were used. An important problem in the clinical application of biomimetic biomaterials with BMPs and/or other morphogens is sterilization. Although gas (ethylene oxide) is used, one always should be concerned about reactive free radicals. Using allogeneic demineralized bone matrix with endogenous native BMPs, as long as low temperature (4C or less) is maintained, the samples tolerated up to 57 M Rads of irradiation. 45,46 The standard dose acceptable to the Food and Drug Administration is 2.5 M Rads. This inform ation would be useful to the biotechnolog y companies preparing to market recombinant BMP-based osteogenic devices. Perhaps, the tissue banking industry with interest in bone grafts 49 could also use this critical information. Various types of freeze-dried and demineralized allogeneic bone may be used in the interim as satisfactory carriers for BMPs. The conclusion of this experiment is that it is not the irradiation dose but the temperature of the sample during irradiation is critical.
MORPHOGENESIS AND TISSUE ENGINEERING and stem cells from marrow and muscle, and a biomaterial scaffolding may lead to an optim al tissue-engineered articular cartilage.
FUTURE CHALLENGES
The symbiosis of biotechnolog y and biomaterials has set the stage for systematic advances in tissue engineering. 55 57 The recent advances in enabling platform technology includes molecular imprinting. 58 In principle, specific recognition and catalytic sites are imprinted using templates. The applications range from biosensors, catalytic applications to antibody, and receptor recognition sites. For example, the cell binding RGD site in fibronectin 59 or YIGSR domain in laminin can be imprinted in complementary sites.60 Let us imagine the tissue engineering of a head of the femur. A mold is fabricated with computer-assisted design and manufacture. It faithfully reproduces the structural features of the femur and may be imprinted with morphogens, inductive signals, and cell adhesion sites. This assembly can be loaded with stem cells and BMPs with a nutrient medium to form new bone in the shape of femoral head. In fact, such a biological approach with vascularized muscle flap and BMPs yielded new bone with a defined shape 61 and is proof of principle for further developm ent and validation. Mikos has developed exciting new injectable polymers such as polypropylene fumarate for tissue engineering of bone leading to much optimism.62 We indeed are entering a brave new world of prefabricated biological spare parts for the human body based on sound architectural rules of inductive signals for morphogenesis, responding stem cells with lineage control, and with growth factors immobilized on a template of biomimetic biomaterial based on extracellular matrix chemistry.
ACKNOWLEDGMENTS
This work is supported by the Lawrence Ellison Chair in Musculoskeletal Molecular Biology. I thank Mrs. Lana Rich and Mrs. Rita Rowlands for their enthusiastic help.
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