TISSUE ENGINEERING Volume 6, Number 4, 2000 Mary Ann Liebert, Inc.

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.

BONE MORPHOGENETIC PROTEINS

Bone grafts have been used by orthopedic surgeons to aid in recalcitrant bone repair for many years. Decalcified bone implants have been used to treat patients with osteomyelitis. 1 Lacroix hypothesize d that bone contains a substance, osteogenin, that initiates bone growth. 2 Urist made the key discovery that demineralized, lyophilized segments of rabbit bone when implanted intramuscularly induced new bone formation. 3 Bone induction is a sequential multistep cascade4 6 that mimics embryonic bone morphogenesis. The key steps in this cascade are chemotaxis, mitosis, and differentiation. Chemotaxis is the directed migration of cells in response to a chemical gradient of signals released from the insoluble demineralized bone matrix. The demineralized bone matrix is predominantly composed of type I insoluble collagen and it binds plasma fibronectin. 7 Fibronectin has domains for binding to collagen, fibrin, and heparin. The responding mesenchymal cells attached to the collagenous matrix and proliferated, as indicated by [3 H]thymidine autoradiography and incorporation into acid-precipitable DNA 8 on day 3. Chondroblast differentiation was evident on day 5, chondrocytes on days 78, and cartilage hypertrophy on day 9. There was concomitant vascular invasion on day 9 with osteoblast differentiation. On days 1012 alkaline phosphatase was maximal. Osteocalcin, bone g -carboxyglut amic acid containing gla protein (BGP), increased on day 28. Hematopoietic marrow differentiated in the ossicle and was maximal by day 21. This entire sequential bone development cascade is reminiscent of cartilage and bone morphogenesis in the limb bud. 5,8 Hence, it has immense implications for isolation of inductive signals initiating cartilage and bone morphogenesis . In fact, a systematic investigation of the chemical components responsible for bone induction was undertaken and inductive signals were identified and isolated successfully.

BIOASSAY FOR BONE MORPHOGENESIS

The above account of the demineralized bone matrix-induced bone morphogene sis in extraskeletal sites demonstrated the possible presence of morphogens in the bone matrix. Thus, a systematic study of the isolation of putative morphogenetic protein was initiated. A prerequisite for any quest for novel morphogens is the establishment of a battery of bioassays for bone formation. A panel of in vitro assays were established for chemotaxis, mitogenesis, and chondrogene sis, and an in vivo assay for osteogenesis. Although the in vitro assays are expedient, we monitored routinely a labor-intensive in vivo bioassay. Furtherm ore, it is the only bona fide bone induction assay. 352

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

PURIFICATION AND CLONING OF BMPS

To scale the procedure up, a switch was made to bovine bone. Demineralized bovine bone was not osteoinductive in rats, and the results were variable. However, when the guanidine extracts of demineralized bovine bone were fractionated on a S-200 molecular sieve column, fractions containing proteins less than 50 kD were consistently osteogenic when bioassayed after reconstitution with allogeneic insoluble collagen. 13,14 Thus, fractions inducing bone were not species specific and are homologous among mammals. It is likely that larger molecular mass fractions and/or the insoluble xenogeneic (bovine and human) collagens were inhibitory or immunogenic. Initial estimates revealed that 1 m g of active osteogenic fraction is present in a kilogram of bone. Hence, over a ton of bovine bone was processed to yield optimal amounts for amino acid sequence determination. The amino acid sequences revealed homology to transforming growth factor-b 1 (TGF-b 1).14 The incisive work of Wozney and colleagues cloned BMP-2, BMP-2B (now called BMP-4), and BMP-3 (also called osteogenin). Osteogenic protein-1 and -2 (OP-1 and OP-2) were cloned by Ozkaynak and colleagues. 12 There are nearly 15 members of the BMP family. The other members of the extended TGF- b /BMP superfamily include inhibins and activins (implicated in follicle-stimulating hormone release from pituitary and mesoderm induction), Mllerian duct inhibitory substance (MIS), growth/differentiation factors (GDFs), and nodal. 14 BMPs are dimeric molecules and the conformation is critical for biological actions. Reduction of the single intermolecular disulfide bond resulted in the loss of biological activity. The mature monom er of BMPs consists of about 120 amino acids, with seven canonical cysteine residues. There are three intrachain disulfides and one interchain disulfide bond. The cysteine knot is the critical central core of the BMP molecule. The crystal structure of BMP-7 has been determined.15 The BMP-7 monomer has b -pleated sheets in the form of two pointed fingers. In the dimer, the pointed fingers are oriented in opposite directions. It is a good possibility in the very near future, based on ligandreceptor cocrystallography, that the receptor contact domains will be defined. Such information will speed up the approaches to design and synthesize peptidomimetic BMPs by combinatorial library techniques using robotic, high-throughpu t assays. Other innovative approaches include screening for small molecules in natural products and biomimetics and receptor-based assays.

CARTILAGE-DERIVED MORPHOGENETIC PROTEINS

Morphogene sis of the cartilage is the key rate-limiting step in the dynamics of bone developm ent. Cartilage is the initial model for the architecture of bones. Bone can form either directly from mesenchyme, as in intramembranous bone formation observed in limited craniofacial bones, or with an intervening cartilage stage, as in endochondra l bone development. 5 All BMPs induce, first, the cascade of chondrogenesis, and, therefore, are cartilage morphogene tic proteins. The hypertrophic chondrocytes in the epiphyseal growth plate mineralize and serve as a template for appositional bone morphogenesis . Cartilage morphogenesis is critical for both bone and joint morphogenesis. The two lineages of cartilage are clear-cut. The first at the ends of bone forms articulating articular cartilage. The second is the grow th plate chondrocyte s, which proliferate, mature, and hypertrophy, synthesize cartilage matrix destined to calcify prior to act as a 353

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

BMPS: PLEIOTROPY AND THRESHOLDS

Morphogene sis is a sequential multistep cascade. BMPs regulate each of the key steps: chemotaxis, mitosis, and differentiation of cartilage and bone. BMPs initiate chondrogene sis in the limb.18 The apical ectodermal ridge is the source of BMPs in the developing limb bud. The intricate dynamic and reciprocal interactions between the epithelium and mesenchyme set into motion the train of events culminating in the pattern formation of digits, radius/ulna, and the humerus in the forelimbs. The chemotaxis of human monocytes is optim al at femtomolar concentration. 19 The apparent affinity was 100200 pM. The mitogenic response was optim al at 100 pM range. The initiation of differentiation was in nanomolar range in solution. However, caution should be exercised because BMPs may be sequestered by extracellular matrix components and the local concentration may be higher when BMPs are bound on the extracellular matrix. Thus, BMPs are pleiotropic regulators that act in a concentration-depende nt thresholds.

BMPS BIND TO EXTRACELLULAR MATRIX

It is well known that extracellular matrix components play a critical role in morphogenesis . The structural macromolecules and their supramolecular assembly in the extracellular matrix do not explain their role in epithelialmesenchymal interactions and morphogenesis. This riddle can now be explained by the binding of BMPs to heparan sulfate, heparin, and type IV collagen 20 of the basement membranes. In fact, this might explain in part the necessity for angiogenesis and vascular invasion into cartilage prior to osteogenesis during development. The actions of activin in development, in terms of dorsal mesoderm induction, are modified to neuralization by binding and termination of activin action by follistatin. 21 Similarly, Chordin and Noggin proteins from the Spemann organizer induce neuralization by binding and inactivation of BMP4. Thus, neural induction is likely to be a default pathway when BMP-4 is rendered nonfunctiona l. 22,23 An emerging principle in developm ent and morphogenesis is that binding proteins can terminate a dominant morphogens action and initiate a default developmental pathway. Furthermore, the binding of a soluble morphogen to extracellular matrix converts it into an insoluble matrix-bound morphogen that can act locally in the solid state 20 and may protect it from proteolysis and prolong its half-life. In this sense, extracellular matrix is both structural and functional as a delivery system for morphogens.

BMPS: ACTIONS BEYOND BONE

Although BMPs were isolated and cloned from bone, recent work with gene knockouts has revealed a plethora of actions beyond bone. Targeted disruption of BMP-2 in mice caused embryonic lethality. Heart development is abnorm al, indicating a need for BMP-2 in heart developm ent.24 BMP-4 knockouts exhibit no mesoderm induction, and gastrulation is impaired. 25 Transgenic overexpression of BMP-6 under the control of the keratin-10 promoter leads to psoriasis. The targeted deletion of BMP-7 revealed the crit354

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.

BMP RECEPTOR KINASES AND SMADS

Recombinant human BMP-4 binds to two type I receptors, BMPR-IA and BMPR-IB, called ALK-3 and ALK-6, respectively.28 BMP-2, BMP-7, and CDMP-1 (GDF-5) bind to both BMPR-IA and IB. There are two types of BMP receptors, types I and II. Both the receptors are membrane-bound serine/threonine kinases. The type II receptors phosphorylate type I receptor. The BMP type I receptor kinases phosphorylat e the Smads.29 Smads are related to the Drosophila Mad (mothers against dpp) gene and three related nematode genes Sma-2, -3, and -4. The terms Sma and Mad have been fused as Smad to unify the nomenclature. There are eight members in the Smad family, which is growing. Phosphorylat ed Smads -1, -5, and -8 are functional mediators of BMP family signaling in partnership with Smad-4. Smads-2 and -3 are signal transducers for actions of TGF-b and activins. Smad-6 and Smad-7 function as antagonists to inhibit TGF- b /BMP superfamily signaling. The phosphorylat ed Smad-1 enters as a heteromeric complex with Smad-4 into the nucleus and activates transcription of early-response genes.30 The BMP receptors also appear to signal via the mitogen-activated protein (MAP) kinase. 31 It is likely that BMPs regulate cell cycle progression and thus govern differentiation of mesenchymal stem cells.

RESPONDING STEM CELLS

It is well known that the embryonic mesoderm-derived mesenchymal cells are progenitors for bone, cartilage, tendons, ligaments, and muscle. However, certain stem cells in adult bone marrow, muscle, and fascia can form bone and cartilage. The identification of stem cells readily sourced from bone marrow may lead to banks of stem cells for cell therapy and perhaps gene therapy with appropriate homing characteristics to bone marrow and hence to the skeleton. The pioneering work of Friedenstein and Owen identified bone marrow stromal stem cells.34,35 These stromal cells are distinct from the hematopoietic stem cell lineage. The bone marrow stromal stem cells consist of inducible and determined osteoprogenitors committed to osteogenesis. Determined osteogenic precursor cells have the propensity to form bone cells without any external cues or signals. On the other hand, inducible osteogenic precursors require an inductive signal such as BMP or demineralized bone matrix. It is noteworthy that operational distinctions between stromal stem cells and hematopoietic stem cells are getting more and more blurry! The stromal stem cells of Friedenstein and Owen are also called mesenchymal stem cells,36,37 with potential to form bone, cartilage, adipocytes, and myoblasts in response to cues from environment and/or intrinsic factors. There is very recently considerable hope and anticipation that these bone marrow stromal cells may be excellent vehicles for cell and gene therapy. From a practical standpoint, these stromal stem cells can be obtained by bone marrow biopsies and expanded rapidly for use in cell therapy after pretreatment with BMPs. The potential uses in both cell and gene therapy are very promising. There are continuous improvements in the viral vectors and efficiency of gene therapy. 38,39 For example, it is possible to use BMP genes transfected in stromal stem cells to target to the bone marrow.

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.

TISSUE ENGINEERING OF CARTILAGE

Unlike bone with its considerable prowess for repair and even regeneration, cartilage is recalcitrant and the biological basis is not clear. In part, this may be due to relative avascularity of hyaline cartilage, high concentration of protease inhibitors, and possibly even endogenous grow th inhibitors in cartilage. Although cartilage has been successfully engineered to predetermined shapes, 50 true repair of the tissue continues to be a real challenge in part due to hierarchical organization and geometry of articular cartilage.51 However, considerable excitement in the field has been generated by a group of Swedish workers, using autologous culture-expanded human chondrocytes.52 These scientists recovered a biopsy of articular cartilage from a non-weight-bearing region and propagated the chondrocyte s rapidly for use in cell therapy. A continuous challenge in chondrocyte cell therapy is progressive dedifferentiation and loss of characteristic cartilage phenotype. The redifferentiation and maintenance of the chondrocyte s for cell therapy can be aided by BMPs, CDMPs, TGF- b isoforms, and IGFs. It is also possible to repair cartilage using muscle-derived mesenchymal stem cells.53 The potential possibility of the problems posed by cartilage proteoglyca ns in preventing cell immigration for repair was investigated by chondroitina se ABC and trypsin pretreatment in partial-thickness defects,54 with and without TGF- b . Pretreatment with chondroitinas e ABC followed by TGF- b revealed a contiguous layer of cells from the synovial membrane hinting at the potential source of repair cells from synovium. Multiple avenues of cartilage morphogens, cell therapy with chondrocyte s 356

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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