Morphogenesis is the developmental cascade of pattern formation, establishment of body plan, and the architecture of mirror-image bilateral symmetry of musculoskeletal structures culmi- nating in the adult form. Regenerative medi- cine is the emerging discipline of the fabrication of spare parts for the human body including the skeleton to restore function of lost parts caused by disease cancer and trauma.
It is based on rational principles of develop- mental biology and morphogenesis. The three key ingredients for regenerative medicine and tissue engineering are inductive morpho- genetic signals, responding stem cells, and extracellular matrix scaffolding.1 Recent advances in molecular cell biology of morpho- genesis will aid in the design principles and architecture for tissue engineering and regen- eration. Regeneration recapitulates embryonic development and morphogenesis. Among the many tissues in the human body, bone has con- siderable powers for regeneration; therefore, it is a prototype model for tissue engineering.
Implantation of demineralized bone matrix into subcutaneous sites results in local bone induction. The sequential cascade of bone mor- phogenesis mimics sequential skeletal mor- phogenesis in limbs and permits the isolation of bone morphogens. Although it is traditional to study morphogenetic signals in embryos and bone morphogenetic proteins (BMPs), the pri- mordial inductive signals for bone are isolated from demineralized bone matrix from adults.
BMPs initiate, promote, and maintain chon- drogenesis and osteogenesis and have actions beyond bone. The recently identified cartilage-
derived morphogenetic proteins (CDMPs) are critical for cartilage and joint morphogenesis.
The symbiosis of bone inductive and conduc- tive strategies is critical for regeneration of bone, and is in turn governed by the context and biomechanics. The context is the microen- vironment, consisting of extracellular matrix scaffolding, and can be duplicated by bio- mimetic biomaterials such as collagens, hydroxyapatite, proteoglycans, and cell adhe- sion proteins including fibronectins and laminins. The rules of architecture for tissue engineering are an imitation and adoption of the laws of developmental biology and mor- phogenesis, thus they may be universal for all tissues, including bones, joints, and associated musculoskeletal tissues.
Bone Morphogenetic Proteins
Bone grafts have been used by orthopedic sur- geons for nearly a century to aid in recalcitrant bone repair. Decalcified bone implants have been used to treat patients with osteomyelitis.2It was hypothesized that bone might contain a sub- stance, osteogenin, that initiates bone growth.3 Urist4 made the key discovery that demineral- ized, lyophilized segments of rabbit bone induced new bone formation when implanted intramuscularly. Bone induction is a sequential multistep cascade.5–7 The key steps in this cas- cade are chemotaxis, mitosis, and differentia- tion. Chemotaxis is the directed migration of cells in response to a chemical gradient of signals
13
Bone Regeneration
A.H. Reddi
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released from the insoluble demineralized bone matrix. The demineralized bone matrix is pre- dominantly composed of type I insoluble colla- gen and it binds plasma fibronectin.8Fibronectin has domains for binding to collagen, fibrin, and heparin. The responding mesenchymal cells attached to the collagenous matrix and prolifer- ated as indicated by [3H]thymidine autoradiog- raphy and incorporation into acid-precipitable DNA on day 3.9Chondroblast differentiation was evident on day 5, chondrocytes on days 7–8, and cartilage hypertrophy on day 9. There was con- comitant vascular invasion on day 9 with osteoblast differentiation. On days 10–12, alka- line phosphatase was maximal. Osteocalcin, bone γ-carboxyglutamic acid containing gla pro- tein (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 bone and cartilage morphogenesis in the limb bud.7,10 Hence, it has immense implications for isolation of inductive signals initiating cartilage and bone morphogenesis. In fact, a systematic investiga- tion of the chemical components responsible for bone induction was undertaken.
A prerequisite for any quest for novel mor- phogens is the establishment of a battery of bioassays for new bone formation. A panel of in vitro assays was established for chemotaxis, mitogenesis, and chondrogenesis, and an in vivo bioassay for bone formation. Although the in vitro assays were expedient, we routinely monitored a labor-intensive in vivo bioassay as it was the only bona fide bone induction assay. A major limitation in the approach was that the demineralized bone matrix is insoluble and in the solid state. In view of this, dissociative extrac- tants such as 4 M guanidine HCl or 8 M urea as 1% sodium dodecyl sulfate at pH 7.4 were used11 to solubilize proteins. Approximately 3% of the proteins were solubilized from demineralized bone matrix, and the remaining residue was mainly insoluble type I bone collagen. The extract alone, or the 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 collaboration between a soluble signal in the extract and insoluble substratum.12This bioassay was a useful advance in the final purification of BMPs and led to the determination of limited
tryptic peptide sequences, which led to the even- tual cloning of BMPs.13–15
The cloning of BMP-2, BMP-2B (now called BMP-4) and BMP-3 (also called osteogenin) by Wozney and colleagues13 was important.
Osteogenic protein-1 and −2 (OP-1 and OP-2) were cloned by Ozkaynak and colleagues.15 There are more than 15 members of the BMP family (Table 13.1). The other members of the extended transforming growth factor β (TGFβ)/BMP superfamily include inhibins and activins (implicated in follicle-stimulating hor- mone release from pituitary) Müllerian duct inhibitory substance, growth/differentiation fac- tors (GDFs), nodal, and lefty, a gene implicated in establishing right/left asymmetry.16,17 BMPs are also implicated in embryonic induction.1,18–20 BMPs are dimeric molecules and the confor- mation is critical for biological actions.
Reduction of the single interchain disulfide bond resulted in the loss of biological activity.
The mature monomer molecule consists of about 120 amino acids, with seven canonical cysteine residues. There are three intrachain disulfides per monomer and one interchain disulfide bond in the dimer. The cysteine knot is in the critical core of the BMP monomer. The crystal structure of BMP-7 has been deter- mined.21 It is a good possibility in the near future that the crystal structure of the BMP-
Table 13.1. Bone morphogenetic proteins
BMP Other names Function
BMP-2 BMP-2A Bone and cartilage morphogenesis BMP-3 Osteogenin Bone morphogenesis BMP-3B GDF-10 Intramembranous bone
formation
BMP-4 BMP-2B Bone formation
BMP-5 Bone morphogenesis
BMP-6 Cartilage hypertrophy
BMP-7 Osteogenic protein-1 Bone formation BMP-8 Osteogenic protein-2 Bone formation
BMP-8B Spermatogenesis
BMP-9 Liver differentiation
BMP-10 ?
BMP-11 GDF-11 ?
CDMP-1 GDF-5 Chondrogenesis, joint formation
CDMP-2 GDF-6 Chondrogenesis
CDMP-3 GDF-7 Tendon/ligament formation
receptor and receptor contact domains will be determined.
BMPs Bind to Extracellular Matrix
It is well known that extracellular matrix compo- nents have a critical role in morphogenesis. The structural macromolecules and their supramol- ecular assembly in the matrix do not explain their role in epithelial–mesenchymal interaction and morphogenesis. This riddle can now be explained by the binding of BMPs to heparan sulfate heparin, and type IV collagen22–24 of the basement membranes. In fact, this might explain in part the necessity for angiogenesis before osteogenesis during development. In addition, the actions of activin in the development of the frog, in terms of dorsal mesoderm induction, are modified to neuralization by follistatin.25 Similarly, Chordin and Noggin from the Spemann organizer induce neuralization by binding and inactivation of BMP-4. Neural induction is likely to be a default pathway when BMP-4 is nonfunctional.26,27 Thus, this is an emerging principle in development and morpho- genesis that binding proteins can terminate a dominant morphogen’s action and initiate a default pathway. Finally, the binding of a soluble morphogen to extracellular matrix converts it into an insoluble matrix-bound morphogen to act locally in the solid state.22
BMP Receptors
Recombinant human BMP-4 and BMP-7 bind to BMP receptor IA (BMPR-IA) and BMP receptor IB (BMPR-IB).28CDMP-1 also binds to both type I BMP receptors. There is a collaboration between type I and type II BMP receptors.29The type I receptor serine/threonine kinase phos- phorylates a signal-transducing protein sub- strate called Smad 1 or 5.30 Smad is a term derived from fusion of Drosophila Mad gene and Caenorhabtitis elegans (nematode) Sma gene. Smads 1 and 5 signal in partnership with a common co-Smad, Smad 4. The transcription of BMP response genes are initiated by Smad 1/Smad 4 heterodimers. Smads are trimeric molecules as gleaned by X-ray crystallography.
The phosphorylation of Smads 1 and 5 by type I
BMP receptor kinase is inhibited by inhibitory Smads 6 and 7.31Smad interacting protein may interact with Smad 1 and modulate BMP response gene expression.15,32The downstream targets of BMP signaling are likely to be home- obox genes, the cardinal genes for morphogene- sis and transcription. BMPs, in turn, may be regulated by members of the hedgehog family of genes such as Sonic and Indian hedgehog33 including receptors patched and smoothened and transcription factors such as Gli 1, 2, and 3.
The actions of BMPs can be terminated by spe- cific binding proteins such as noggin.26
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 mar- row, muscle, and fascia can form bone and car- tilage. 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” characteris- tics to bone marrow and hence to the skeleton.
The pioneering work of Friedenstein et al.34and Owen and Friedenstein35 identified bone mar- row stromal stem cells. These stromal cells are distinct from the hematopoietic stem cell line- age. 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. However, inducible osteogenic precur- sors require an inductive signal such as BMP or demineralized bone matrix. The stromal stem cells of Friedenstein and Owen are also called mesenchymal stem cells,36 with potential to form bone, cartilage, adipocytes, and myoblasts in response to cues from environment and/or intrinsic factors. There is very recently consider- able hope and anticipation that these bone mar- row stromal cells may be excellent vehicles for cell and gene therapy.37
From a practical standpoint, these stromal stem cells can be obtained by bone marrow biop- sies 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.38,39It is possible to use BMP genes transfected in stro- mal stem cells to target to the bone marrow to induce bone.40
Morphogens and Gene Therapy
The recent advances in morphogens are ripe for techniques of regional gene therapy for orthope- dic tissue engineering. The availability of cloned genes for BMPs and CDMPs and the requisite platform technology of gene therapy may have immediate applications. Whereas protein ther- apy provides an immediate bolus of morphogen, gene therapy achieves a sustained prolonged secretion of gene products. Furthermore, recent improvements in regulated gene expression allow the turning on and off of gene expression.
The progress in vectors for delivering genes also bodes well. The use of adenoviruses, adeno- associated viruses, and tetroviruses is poised for applications in bone and joint repair.38,39,41,42
Although gene therapy has some advantages for orthopedic tissue engineering, an optimal deliv- ery system for protein and gene therapy is needed, especially in replacement of large seg- mented defects and in fibrous non-unions and malunions.
Challenges
The earlier discussion of inductive signals (BMPs), responding stem cells (stromal cells) leads us to the scaffolding (the microenviron- ment/extracellular matrix) for optimal tissue engineering. The natural biomaterials in the composite tissue of bones and joints are colla- gens, proteoglycans, and glycoproteins of cell adhesion such as fibronectin and the mineral phase. The mineral phase in bone is predomi- nantly hydroxyapatite. In native state, the asso- ciated citrate, fluoride, carbonate, and trace elements constitute the physiological hydroxya- patite. The high protein binding capacity makes hydroxyapatite a natural delivery system.
Comparison of insoluble collagen, hydro- xyapatite, tricalcium phosphate, glass beads, polymethylmethacrylate as carriers, revealed collagen to be an optimal delivery system for BMPs.43It is well known that collagen is an ideal
delivery system for growth factors in soft and hard tissue wound repair.44
During the course of systematic work on hydroxyapatite of two pore sizes (200 or 500 µm) in two geometrical forms (beads or discs), an unexpected observation was made. The geome- try of the delivery system is critical for optimal bone induction. The discs were consistently osteoinductive with BMPs in rats, but the beads were inactive.45The chemical composition of the two hydroxyapatite configurations was identical.
In certain species, the hydroxyapatite alone appears to be “osteoinductive.”46In subhuman primates, the hydroxyapatite induces bone, albeit at a much slower rate. One interpretation is that osteoinductive endogenous BMPs in cir- culation progressively bind to implanted discs of hydroxyapatite. When an optimal threshold con- centration of native BMPs is achieved, the hydroxyapatite becomes osteoinductive. Strictly speaking, most hydroxyapatite substrata are ideal osteoconductive materials. This example in certain species also serves to illustrate how an osteoconductive biomimetic biomaterial may progressively function as an osteoinductive sub- stance by binding to endogenous BMPs. Thus, there is a physiological–physicochemical contin- uum between the hydroxyapatite alone and a progressive composite with endogenous BMPs.
Recognition of this experimental nuance will save unnecessary arguments among biomaterials scientists about the osteoinductive action of a conductive substratum such as hydroxyapatite.
Complete regeneration of a baboon cran- iotomy defect was accomplished by recombi- nant human osteogenic protein (rhOP-1; human BMP-7).47Recombinant BMP-2 was delivered by poly(α-hydroxy acid) carrier for calvarial regen- eration.48Copolymer of polylactic and polygly- colic acid in a non-union model in the rabbit ulna was used and the results were satisfactory.49 An important problem in the clinical applica- tion of biomimetic biomaterials with BMPs and/or other morphogens is the sterilization.
Although gas (ethylene oxide) is used, one should always be concerned about reactive free radicals. Using allogeneic demineralized bone matrix with endogenous native BMPs, as long as low temperature (4˚C or less) was maintained, the samples tolerated up to 5–7 M Rads of irradiation.50,51The standard dose acceptable to the Food and Drug Administration is 2.5 M Rads. This information would be useful to the biotechnology companies preparing to market
recombinant BMP-based osteogenic devices.
Perhaps the tissue banking industry with an interest in bone grafts52could also use this criti- cal information. The various freeze-dried and demineralized allogeneic bone may be used in the interim as satisfactory carriers for BMPs.
The moral of this experiment is it is not the irra- diation dose, but the ambient sample tempera- ture during irradiation that is absolutely critical.
Unlike bone with its considerable prowess for repair and even regeneration, cartilage is recal- citrant. But why? It may be attributable in part to relative avascularity of hyaline cartilage, high concentration of protease inhibitors, and per- haps even growth inhibitors. The wound debridement phase is not optimal to prepare the cartilage wound bed for the optimal milieu interieur for repair. Although cartilage has been successfully engineered to predetermined shapes,53 true repair of the tissue continues to be a real challenge in part because of hierarchi- cal organization and geometry.54However, con- siderable excitement in the field has been generated by a group of Swedish workers in Göteborg, using autologous culture-expanded human chondrocytes.55A continuous challenge in chondrocyte cell therapy is progressive ded- ifferentiation and loss of characteristic cartilage phenotype. The redifferentiation and mainte- nance of the chondrocytes for cell therapy can be aided by BMPs, CDMPs, TGFβ isoforms, and insulin-like growth factors. It is also possible to repair cartilage using muscle-derived mes- enchymal stem cells.56The potential possibility of the problems posed by cartilage proteogly- cans in preventing cell immigration for repair was investigated by chondroitinase ABC and trypsin pretreatment in partial-thickness defects,57with and without TGFβ. Pretreatment with chondroitinase ABC followed by TGFβ 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 chondrocytes and stem cells from marrow and muscle, and biomaterial scaffold- ing may lead to an optimal tissue engineered articular cartilage.
It is inevitable during the aging of humans that one will confront impaired locomotion caused by wear and tear in bones and joints.
Therefore, the repair and possibly complete regeneration of the musculoskeletal system and other vital organs such as skin, liver, and kidney
may potentially need optimal repair or a spare part for replacement. Can we create spare parts for the human body? There is much reason for optimism that tissue engineering can help patients. We are living at an extraordinary time in the biology, medicine, surgery, and computa- tional and related technology. The confluence of advances in molecular developmental biology and attendant advances in inductive signals for morphogenesis, stem cells, biomimetic biomate- rials, and extracellular matrix biology augers well for imminent breakthroughs.
The symbiosis of biotechnology and bioma- terials has set the stage for systematic advances in tissue engineering.58–60 The recent advances in enabling platform technology include molec- ular imprinting.61In principle, specific recogni- tion and catalytic sites are imprinted using templates. The applications range from biosen- sors, catalytic applications to antibody, and receptor recognition sites. For example, the cell binding RGD site in fibronectin62 or YIGSR domain in laminin can be imprinted in comple- mentary sites.63
The rapidly advancing frontiers in morpho- genesis with BMPs, hedgehogs, homeobox genes, and a veritable cornucopia of general and specific transcription factors, coactivators, and repressors will lead to cocrystallization of ligand–receptor complexes, protein–DNA complexes, and other macromolecular interac- tions. Let us imagine a head of the femur and a mold is fabricated with computer-assisted design and manufacture. It faithfully repro- duces the structural features and may be imprinted with morphogens, inductive signals, and cell adhesion sites. This assembly can be loaded with stem cells and BMPs and other inductive signals with a nutrient medium opti- mized for optimal number of cell cycles, and then predictably exit into the differentiation phase to reproduce a totally new bone femoral head. In fact, such a biological approach with vascularized muscle flap and BMPs yielded new bone with a defined shape and has demon- strated the proof of principle for further devel- opment and validation.64 We indeed are entering a brave new world of prefabricated biological spare parts for the regeneration of bone for the human body based on sound architectural rules of inductive signals for mor- phogenesis, responding stem cells with lineage control, and with growth factors immobilized on a template of biomimetic biomaterial based
on extracellular matrix. The BMPs are now approved by the Food and Drug Adminis- tration for clinical uses in spine fusion and fracture healing.
Acknowledgment
This work was supported by the Lawrence Ellison Chair in Musculoskeletal Molecular Biology.
I thank Ms. Danielle Neff for outstanding help and enthusiastic bibliographic assistance.
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