Cellular Strategies for Fracture Repair

COPYRIGHT 2008 BY THE JOURNAL OF BONE AND JOINT SURGERY, INCORPORATED

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Cellular Strategies for Enhancement of Fracture Repair

By Thomas E. Patterson, PhD, Ken Kumagai, MD, PhD, Linda Griffith, PhD, and George F. Muschler, MD

Tissue engineering seeks to translate scientific knowledge into tangible products to advance the repair, replace-ment, or regeneration of organs and tissues. Current tissue engineering strategies have progressed recentlyfrom a historical approach that is based primarily on biomaterials to a cell and tissue-based approach that in-cludes understanding of cell-sourcing and bioactive stimuli. New options include methods for harvest and trans-plantation of tissue-forming cells, bioactive matrix materials that act as tissue scaffolds, and delivery ofbioactive molecules within scaffolds. These strategies are already benefiting patients, and they place increasingdemands on orthopaedic surgeons to have a solid foundation in the contemporary concepts and principles ofcell-based tissue engineering.

Essentially all orthopaedic tissue engineering strategies can be distilled to a strategy or combination of strategiesthat seek to increase the number or relative performance of bone-forming cells. The global term connective tissueprogenitors has been used to define the heterogeneous populations of stem and progenitor cells that are found innative tissue and that are capable of differentiating into one or more connective tissue phenotypes. These stem orprogenitor populations are found in various tissue sources, with varying degrees of ability to differentiate alongconnective tissue lineages. Available cell-based strategies include targeting local cells with use of scaffolds or bio-active factors, or transplantation of autogenous connective tissue progenitor cells derived from bone marrow orother tissues, with or without processing to change their concentration or prevalence. The future may includemeans of homing circulating connective tissue progenitor cells with use of intrinsic chemokine systems, or modify-ing the biological performance of connective tissue progenitor cells by means of genetic modifications.

Stem and Progenitor Cells in Musculoskeletal Tissues

tem and progenitor cells are present in all adult tissuesand are critical to tissue health, maintenance, and re-sponse to injury or disease throughout life. Stem cells

give rise to progenitor cells and are distinguished from themby their capacity for self-renewal by a process of asymmetriccell division. Progenitor cells, by definition, have finite limitson their capacity for self-renewal and generally progress togive rise to one or more differentiated phenotypes1,2.

Stem and progenitor cell populations are the upstreamcomponents of continuous systems of cell renewal in virtuallyall human tissues. This turnover is most evident in tissues thatremodel rapidly, such as the lining cells of the gastrointestinaltract (replaced every three days) or dermis (replaced every twoweeks). In bone, turnover is much slower. Osteocytes or bone-

lining cells, the differentiated cells that define bone tissue, maysurvive for twenty years in human cortical bone. However,continuous remodeling requires the formation of many newosteoblasts. Osteoblasts, in turn, are continuously derivedfrom a much smaller number of preosteoblasts and upstreamprogenitor cells. The number of true stem cells needed to sup-port this process may be very small (on the average, less thanone in 20,000 nucleated cells in native marrow). The activa-tion of stem cells and the proliferation of progenitor cells toform new osteoblasts are vastly accelerated as a result oftrauma, such as fractures2.

In the 1960s, Burwell showed that implantation of can-cellous bone grafts induced bone formation, which could betraced to the activity of primitive osteogenic cells in bonemarrow3. Friedenstein4 showed that new bone was formed byproliferating fibroblast-like marrow cells and that the number

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Disclosure: In support of their research for or preparation of this work, one or more of the authors received, in any one year, outside funding orgrants in excess of $10,000 from the National Institutes of Health (the National Institute of Arthritis and Musculoskeletal and Skin Diseases[NIAMS] and the National Institute of General Medical Sciences [NIGMS]) and Therics, Inc. One or more of the authors, or a member of his or herimmediate family, received, in any one year, payments or other benefits or a commitment or agreement to provide such benefits from commercialentities in excess of $10,000 (DePuy, Synthes, and Therics, Inc.) and less than $10,000 (Orthofix). No commercial entity paid or directed, oragreed to pay or direct, any benefits to any research fund, foundation, division, center, clinical practice, or other charitable or nonprofit organizationwith which the authors, or a member of their immediate families, are affiliated or associated.

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of these proliferating cells could be assayed by counting thenumber of fibroblastic colony-forming units in vitro. An in-triguing feature of many tissues, including musculoskeletaltissues, is that upstream progenitor cells are often multipotent.Stem cells derived from bone, bone marrow, and peritrabecu-lar tissues in cancellous bone, periosteum, cartilage, muscle,fat, and vascular pericytes are capable of differentiation intomultiple phenotypes, including bone, cartilage, tendon, liga-ment, fat, muscle, and nerve5-18. This has important implica-tions with regard to the design of tissue engineering strategies,in that cells derived from one tissue might be useful in form-ing other tissue types. Harvest of these tissues varies with re-spect to the associated host morbidity19-25. Aspiration of bonemarrow is associated with the least morbidity and provides asingle-cell suspension that can be readily processed intraoper-atively for immediate implantation13. Fat has also been pro-posed as a low-morbidity tissue source, although it requiresgreater processing11,26. Many names have been used to describethe colony-forming cells found in bone marrow, periosteum,or trabecular bone, in addition to fibroblastic colony-formingunits. These terms include mechanocytes, bone marrow stro-mal cells, and mesenchymal stem cells, although the precisedefinition and biological capabilities ascribed by these termsare not entirely synonymous.

For purposes of describing experimental and clinical re-sults with use of primary cells isolated from human tissue andgrown without culture expansion, we have defined the termconnective tissue progenitor as the entire heterogeneous po-pulation of stem and progenitor cells that are capable of dif-ferentiating into one or more connective tissue phenotypes,including bone, fat, cartilage, blood, and fibrous tissues1. Thenumber of connective tissue progenitor cells in various tissuesis most often estimated with use of the colony-forming unitassays, that is, cells that give rise to a colony of proliferatingprogenitor cells in vitro under conditions that are selected topromote activation and proliferation of one or more fractionsof the connective tissue progenitor population. In these assays,each colony represents the progeny of a founding stem or pro-genitor cell. Functional biological differences between thecolony-founding cells are revealed by differences in the prolif-eration rate, the morphology, the migration, and the differen-tiation of their progeny in each colony (Fig. 1).

The term connective tissue progenitor recognizes thatthese tissue-derived cells are not a pure or uniform popula-tion and may be derived from more than one pool of stemcells and progenitor cells in native tissues. These cells may in-clude true quiescent, multipotent stem cells that become acti-vated after harvest and are capable of self-renewal. However,colonies may also be formed by cells that are already prolifer-ating in vivocells that lack self-renewal capabilities andmay exhibit intrinsic commitment to various stages of diverselineages17,27,28. This diversity can be a source of frustration forthose looking for homogeneous purified populations of cells,but in practice this diversity should be expected, given themultifunctional nature of the bone marrow environment.Therefore, this diversity can also be viewed as a source of

valuable information that can be dissected experimentally tounderstand the prevalence and kinetics of various connectivetissue stem-cell populations and to understand the ways inwhich these populations change according to age, gender, dis-ease states, pharmacological intervention, and tissue engi-neering strategies1,13,29.

Another experimental and tissue engineering approachto study stem and progenitor populations from bone marrowor other tissue sources involves the in vitro expansion of cellsderived from connective tissue progenitor cells. Various nameshave been given to culture-expanded populations of cells thathave been selected under different culture conditions, includ-ing bone marrow stromal cells15, mesenchymal stem cells30,and adult multipotential progenitor cells31,32. These terms arenot synonymous1, but they all denote that progenitor cells canbe isolated and expanded under appropriate conditions andthat these cells can retain the capacity to differentiate into avariety of musculoskeletal phenotypes15,17,30. The term mesen-chymal stem cell has acquired a particularly narrow definitionof bone marrow-derived, culture-expanded cells that are iso-lated and expanded according to the methods pioneered byCaplan33.

Culture-expanded cell populations differ dramaticallyfrom the heterogeneous population of connective tissue pro-genitor cells that are present in native tissue. Under in vitroculture conditions, the heterogeneous population rapidly be-comes more homogeneous. When cells are grown in vitro,clones of cells that divide most rapidly and those that have thegreatest capacity for continued proliferation have a competi-tive advantage. In vitro expansion therefore produces a strongselective pressure favoring these traits.

All tissues vary substantially with respect to cellularityand the prevalence of connective tissue progenitor cells. As-pirated bone marrow is the best characterized source ofconnective tissue progenitor cells, containing a mean of ap-proximately forty million nucleated cells and approximately2000 connective tissue progenitor cells per milliliter. How-ever, the yield of connective tissue progenitor cells can varywidely between individuals, aspiration sites, and even be-tween individual aspirations13,25,29. In contrast, fat is far lesscellular (approximately six million cells per cubic centimeterof tissue) and the prevalence of connective tissue progenitorcells is greater (as high as one per 4000 cells), but fat-derivedconnective tissue progenitor cells exhibit different patterns ofproliferation, migration, and differentiation than do bonemarrow-derived connective tissue progenitor cells.

Differences between connective tissue progenitor cellsharvested from different individuals and from various tissuesources are likely a function of the health and histologicalcharacteristics of the local tissues and reflect the underlyingkinetics of stem cell function in that tissue. These variables arein turn influenced by age, gender, and both local and systemicdisease1,13,29,34. For example, bone marrow cellularity declineswith age. There is also an age-related decline in the prevalenceof connective tissue progenitor cells, at least in women29. How-ever, age and gender account for only a small fraction of the


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variation in the concentration and prevalence of connectivetissue progenitor cells among patients13,25,29. As a result, osteo-genic connective tissue progenitor cells can be harvested withuse of bone marrow aspiration in patients of all ages.

Differences in biological potential among connectivetissue progenitor cells derived from various tissues can haveimportant practical implications with regard to the selectionof cell sources for tissue engineering. Individual bone marrow-derived connective tissue progenitor cells can be capable ofdifferentiating into a broad range of phenotypes, includingbone, fibrous tissue, fat, muscle, cartilage, and perhaps evenneural tissue, liver, and cardiac muscle1,2,15,27,30,35,36. Connectivetissue progenitor cells derived from muscle, fat, and cartilagealso have a broad repertoire of intrinsic differentia-tion11,12,20,21,24,37. Some studies have suggested that fat-derivedand bone marrow-derived cells are similar38, but others havedemonstrated a decreased osteogenic potential in fat-derived

cells39 and the absence of surface markers characteristic of os-teoblastic progenitor cells40.

Potential Strategies for Use of Autogenous Connective Tissue Progenitor Cells in Therapeutic Applications

here are five major types of cell-based tissue engineering:(1) local targeting of connective tissue progenitor cells

where new tissue is needed, (2) homing of connective tissueprogenitor cells into areas where they may not currently re-side, (3) physically transplanting autogenous connective tissueprogenitor cells to augment the local population, (4) trans-planting culture-expanded or modified connective tissue pro-genitor cells, and (5) transplanting fully formed tissue.

Targeting Connective Tissue Progenitors in SituLocal targeting strategies are designed to promote desired tis-

T

Fig. 1

Heterogeneity of connective tissue progenitor cells. The colonies shown in this image were cul-

tured from human bone marrow for nine days and then stained for alkaline phosphatase activity,

a marker of early bone formation. The image illustrates that colonies differ in size, cell density,

and the extent and distribution of alkaline phosphatase activity. These morphologic differences

are manifestations of intrinsic differences among connective tissue progenitor cells at the time

that they were harvested from bone marrow and placed into culture. (Reprinted from: Muschler

GF, Nakamoto C, Griffith LG. Current concepts review. Engineering principles of clinical cell-based

tissue engineering. J Bone Joint Surg Am. 2004;86:1541-58.)


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sue formation by stimulating the activation, migration, pro-liferation, and/or differentiation of local connective tissueprogenitor cells. The strategy relies on a sufficient local popula-tion of connective tissue progenitor cells. Tissue scaffolds pro-vide a surface on which cells and connective tissue progenitorcells can attach, proliferate, migrate, and differentiate. Thesescaffolds also prevent the encroachment of adjacent tissues intoan area where new tissue is desired. Examples of targeting in-clude implantation of acellular tissue scaffolds (e.g., allograftbone); locally delivered growth factors (e.g., bone morphoge-netic proteins); biophysical stimulation, such as mechanicalloading41-43, electromagnetic stimulation44-46, or ultrasound47;and systemic pharmacological strategies (e.g., parathyroid hor-mone for the treatment of osteoporosis48-53 or systemic growthhormones to increase muscle mass in the elderly54,55).

Homing of Connective Tissue Progenitor CellsHoming generally refers to the recruitment of cells from thesystemic circulation. Homing and the underlying mechanismsof homing are well established in hematopoietic connectivetissue progenitor cells. Several studies have suggested that os-teogenic connective tissue progenitor cells may travel throughthe systemic circulation56, although the extent to which circu-lating osteogenic cells contribute to normal fracture repair isonly beginning to be characterized.

With use of a mouse parabiosis model, Kumagai et al.have recently shown that circulating cells do home to the siteof a fibular fracture57. By two weeks after fracture, roughly 6%to 12% of cells that express alkaline phosphatase in the frac-ture sites (interpreted as osteoblasts) were found to be derivedfrom circulating cells57. These and other data suggest that stemcell homing may be a normal biological process that may be-come the target of new therapies to enhance the arrival of os-teogenic connective tissue progenitor cells at sites of bonerepair.

Transplantation of Connective Tissue Progenitor CellsIn many clinical wound-healing scenarios, new tissue forma-tion may be hampered by a local deficiency or suboptimal lo-cal population of connective tissue progenitor cells. This isparticularly true in regions of previous trauma, infection, irra-diation, scar, or compromised vascularity. Many studies haveshown that transplantation of connective tissue progenitorcells into a bone-healing site can improve the outcome of bothconductive and inductive grafts, even in sites that are sur-rounded by nondiseased tissues1,58. This suggests that manyand perhaps all situations of normal tissue repair may be lim-ited by the population of connective tissue progenitor cells inlocal tissues.

Autogenous cancellous bone-grafting has long been themost prevalent and effective example of cell transplantation.Several clinical studies have suggested that transplantation ofconnective tissue progenitor cells in aspirated bone marrowhas value in bone-healing applications3,10,59-65. Additionally,concentration of bone marrow cells by centrifugation could

increase osteogenesis further. Many surgeons now use bonemarrow because of its biological value and low risk.

The aspiration technique is important. Muschler et al.found that limiting the volume of the aspirate to 2 mL persite reduces dilution with peripheral blood and increases theconcentration of marrow-derived connective tissue progenitorcells13. Furthermore, the efficacy of a bone marrow graft canbe enhanced by the use of certain porous implantable materi-als to selectively concentrate and select marrow-derived con-nective tissue progenitor cells from bone marrow, a processknown as selective retention58. Selective retention of connectivetissue progenitor cells can be used to rapidly enrich the popu-lation of marrow-derived connective tissue progenitor cells byremoving red blood cells, serum, and most other cells in mar-row and contaminating cells from peripheral blood. Grafts en-riched in this way have improved the results of bone-graftingin a canine spinal-fusion model58. The use of a centrifuge toconcentrate low-density cells from bone marrow (buffy-coatcells) for transplantation has also been described by bothConnolly66,67 and Hernigou et al.68.

Transplantation of Culture-Expanded CellsCulture-expanded cells from muscle, fat, and bone marrowmay be useful in regeneration of bone, cartilage, muscle, andtendon tissue6,35,69-75. In vitro expansion can generate a largenumber of progenitor cells; however, it also adds substantialcost and some risks, such as contamination with bacteria orviruses or depletion of proliferative capacity76-78. This strategyis already applied clinically in the area of cartilage repair79-81. Invitro selection of the most rapidly proliferating cells may alsoselect cells with mutations or epigenetic changes that mightconfer a tumor-forming potential, although we are not awareof any reports of human tumors formed by culture-expandedcells and the risk of tumor formation appears to be very low.

Transplantation of Genetically Modified Cells and Their ProgenyThe intrinsic biological potential and performance of connec-tive tissue progenitor cells and their progeny can be geneticallymodified by either transiently or permanently altering the ex-pression of one or more genes82. Advances in genetic engineer-ing techniques facilitate the efficient engineering of cells thatsecrete factors (e.g., bone morphogenetic protein-2) that ef-fect a change in local tissue formation83. Although transplanta-tion of genetically modified cells may not play a role in electiveclinical tissue engineering in the near future, it has substantialpotential value, particularly in the setting of inherited geneticdefects (e.g., osteogenesis imperfecta84) and for tissues (such ascartilage) that consist of relatively homogeneous long-livedcells and in which stable phenotypic expression may be a cur-rent limitation in biological outcome85,86.

Mass Transport Limitations and Metabolic Demand

n all cell-transplantation settings, access to substrate mole-cules and clearance of metabolic products are critical to cellI


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survival. Due to high demand and slow diffusion rates for ox-ygen, few cells tolerate diffusion distances in excess of 0.2 mm.At a graft site in which the radius of the graft is approximately5 mm, diffusion of oxygen is able to support only a limitednumber of transplanted cells, resulting in central necrosis.This limitation highlights the need to design bone grafts that(1) limit the total number of cells and (2) increase the fractionof transplanted cells that will contribute to tissue repair, eitherby positive selection or by negative selection (removal of non-contributing, inhibitory, or competing cells).

Design and Selection of Scaffolds for Tissue Engineering

hree-dimensional porous scaffolds can be designed withspecific architectures at the nano, micro, and macro scale

(i.e., molecular, cellular, and tissue-length scales, respectively).Desirable scaffold features include (1) preservation of a tis-sue volume for the formation of new tissue; (2) nanoporos-ity and microporosity that allows effective mass transport tosupport transplanted cells; (3) a connected microporosity andmacroporosity that allows for contiguous tissue and vascularingrowth; (4) a surface chemistry and texture that enhancesthe attachment, migration, proliferation, and differentiationof osteogenic connective tissue progenitor cells; and (5) degra-dation properties that are consistent with preservation of thehealth of local tissues and effective ongoing remodeling. Cur-rent scaffold options include tissue-derived materials, biologi-cal polymers, ceramics or mineral-based matrices, and metalsas well as composites of two or more materials. The overallmechanical properties of a scaffold (strength, modulus, andtoughness) are determined both by the material properties ofthe bulk material and by its three-dimensional structure.Matching the mechanical properties of a scaffold to the graftenvironment is critically important so that progression oftissue-healing is not limited by mechanical failure of the scaf-fold prior to successful tissue regeneration. Rapidly evolvingthree-dimensional fabrication methods (e.g., three-dimen-sional printing and three-dimensional stereolithography) aswell as the development of new materials (e.g., polycarbo-nates87-89 and polypropylene fumarates90-93) provide highlypromising platforms for future development.

The surface chemistry defines much of the environ-ment that cells will experience soon after implantation andhas a profound effect on the attachment and survival of cellsfollowing implantation as well as on early proliferation,migration, and differentiation. Implanted materials rapidlybecome coated with proteins and lipids, which are the prin-cipal mediators of the cellular response to most materials. Ithas been speculated that hydroxyapatite and some other ce-ramics may preferentially sequester bioactive molecules thatare important for bone regeneration. Indeed, hydroxyapa-tite and tricalcium phosphate materials perform successfullyas depot delivery vehicles for bone morphogenetic proteinsboth in animals94 and humans95,96. Protein adsorption can,however, induce a conformational change, which may hideor expose sites that interact with cell-surface receptors. For

example, fibronectin is a more active adhesion moleculeon hydrophilic surfaces (e.g., glass) than on hydrophobicsurfaces (e.g., polytetrafluoroethylene [Teflon] or polyethyl-ene)97-100. The attachment, survival, proliferation, and dif-ferentiation of stem and progenitor cells can be modulatedin vitro if scaffold surfaces are precoated with selected bioac-tive proteins, including bone morphogenetic protein-2 andbone morphogenetic protein-7101-105.

Proteins and small bioactive peptides can also be selec-tively concentrated and presented by covalently linking themto a surface96,106,107. This provides more control over conforma-tion, a slower rate of release from the surface, and longer re-tention. Presentation of growth factors in a matrix-boundfashion may better mimic the native physiology of many sol-uble signaling molecules, including most proteins. Tetheringmay not be appropriate, however, for signaling moleculesthat need to be internalized (e.g., steroid hormones)108-111.This strategy may also be particularly well suited for the de-sign of matrices with selective affinity for specific cells or setsof cells (e.g., connective tissue progenitor cells, endothelialcells, and platelets).

The pharmacokinetics of delivery of bone morphoge-netic proteins has been shown to be an important clinicalvariable in a variety of materials, including degradablepolymers112-119, type-I collagen120-122, and calcium phosphateceramics123,124. The retention time of implanted bone morpho-genetic protein correlates with biological efficacy, presumablybecause the longer a bone morphogenetic protein is retained,the higher the probability that it will act on an appropriatetarget cell. Retention time has been related both to solubility125

and to isoelectric point94.Current clinical strategies for protein delivery are tech-

nically simple but require that the protein be delivered in ahigh concentration in order to diffuse into adjacent tissuesand act on local connective tissue progenitor cells. The disad-vantage of these strategies is that they provide relatively littlecontrol over the rate of delivery, conformation, presentation,clearance, or degradation of the delivered protein. Althoughcurrent strategies for delivery of bone morphogenetic proteinscan be effective, the vast majority of the massively supraphysi-ological doses of bone morphogenetic proteins that are cur-rently required are likely wasted, and only a small fractionactually elicits a receptor-mediated signal that enhances newbone formation. These methods, therefore, leave substantialroom for improvement in delivery kinetics and distribution ofbioactive proteins.

A scaffold designed to deliver viable cells must providean environment with physiologic pH and osmolarity. The de-sign of scaffold bulk material must also consider the effects ofdegradation products. The degradation of many polyester-based matrices, such as polylactides and polyglycolides, pro-duces acidic degradation products (lactic acid and glycolicacid), and therefore those matrices are not ideal for cell trans-plantation and tissue regeneration. Similarly, materials thatresult in an early hyperosmolar environment, such as glycerol(used to improve handling of many bone pastes) and calcium

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sulfate (both acidic and hyperosmolar), are likely not appro-priate materials for use in a cell-transplantation environment.

Opportunities for Rational Design of Future Materials, Devices, and StrategiesOptimizing combinations of cells, matrices, and locally andsystemically active stimuli will remain a complex process char-acterized by a highly interdependent set of variables with analmost infinite range of possible combinations. Future tissueengineering strategies that are particularly rich in opportunityinclude (1) improved methods for intraoperative harvest, con-centration, and selection of stem and progenitor cells; (2) cell-delivery systems that enhance cell survival by managing thebalance of mass transfer and metabolic demand; (3) three-dimensional scaffolds with architectural and mechanicalfeatures that are customized for specific clinical applications;(4) chemically defined surfaces that present covalently teth-ered, biologically active molecules; (5) defined microtexturedsurfaces to elicit desired cell attachment, migration, differenti-ation, and survival; (6) scaffold materials for which degrada-tion delivers biologically inert or even bioactive molecules,minimizing the toxicity associated with degradation prod-ucts; and (7) delivery systems for soluble molecule (e.g., bonemorphogenetic proteins and other protein growth factors)delivery systems that ensure both a biologically active con-formation and provide a local concentration profile that is ap-propriate for the target cell population, minimizing the totaldose of bioactive agent that is required and the attendant riskof unwanted collateral effects.

These developments in cell-based approaches to im-

prove fracture repair must also be informed by a combinationof clinical experience, knowledge of basic biological princi-ples, medical necessity, and commercial practicality. The re-sponsibility for rational development is shared by the entireorthopaedic community (developers, vendors, and physi-cians) and must be focused as much as possible on objectiveand systematic assessment and reporting. This challenge ismade particularly urgent by the recent rapid addition of manynew clinical options that often have narrow and nonoverlap-ping regulatory approval but that also are rapidly applied inoff-label applications by clinicians earnestly seeking the bestpossible care for their patients. Prospective, randomized pre-clinical and clinical trials will continue to play a critical role inthe initial evaluation of new materials for specific indications.In addition, prospective cohort studies will remain critical as ameans of demonstrating that controlled studies can be gener-alized to the broader orthopaedic community. Prospectiveregistries related to trauma care will also play an importantrole by objectively defining settings in which current practicefalls short of reported or desired outcomes. Such registriescould be applied directly to define opportunities for neededprospective trial and identify settings in which randomizationis impractical due to insufficient power or unethical due to ex-isting evidence of success or failure in specific settings.

Corresponding author:George F. Muschler, MDDepartments of Orthopaedic Surgery and Biomedical Engineering (ND-20), Cleveland Clinic, 9500 Euclid Avenue, Cleveland, OH 44195. E-mail address: [email protected]

References

1. Muschler GF, Midura RJ. Connective tissue progenitors: practical concepts for clinical applications. Clin Orthop Relat Res. 2002;395:66-80.

2. Muschler GF, Midura RJ, Nakamoto C. Practical modeling concepts for connec-tive tissue stem cell and progenitor compartment kinetics. J Biomed Biotechnol. 2003;3:170-93.

3. Burwell RG. Studies in the transplantation of bone. VII. The fresh composite homograft-autograft of cancellous bone. An analysis of factors leading to osteo-genesis in marrow transplants and marrow-containing bone grafts. J Bone Joint Surg Br. 1964;46:110-40.

4. Friedenstein AJ. Precursor cells of mechanocytes. Int Rev Cytol. 1976;47:327-59.

5. Brighton CT, Lorich DG, Kupcha R, Reilly TM, Jones AR, Woodbury RA 2nd. The pericyte as a possible osteoblast progenitor cell. Clin Orthop Relat Res. 1992;275:287-99.

6. Caplan AI, Bruder SP. Mesenchymal stem cells: building blocks for molecular medicine in the 21st century. Trends Mol Med. 2001;7:259-64.

7. Connolly J, Guse R, Lippiello L, Dehne R. Development of an osteogenic bone-marrow preparation. J Bone Joint Surg Am. 1989;71:684-91.

8. Dore-Duffy P, Katychev A, Wang X, Van Buren E. CNS microvascular pericytes exhibit multipotential stem cell activity. J Cereb Blood Flow Metab. 2006;26:613-24.

9. Friedenstein AJ, Petrakova KV, Kurolesova AI, Frolova GP. Heterotopic of bone marrow. Analysis of precursor cells for osteogenic and hematopoietic tissues. Transplantation. 1968;6:230-47.

10. Garg NK, Gaur S. Percutaneous autogenous bone-marrow grafting in congeni-tal tibial pseudarthrosis. J Bone Joint Surg Br. 1995;77:830-1. Erratum in: J Bone Joint Surg Br. 1996;78:683-4.

11. Gimble JM, Robinson CE, Wu X, Kelly KA. The function of adipocytes in the

bone marrow stroma: an update. Bone. 1996;19:421-8.

12. Huard C, Moisset PA, Dicaire A, Merly F, Tardif F, Asselin I, Tremblay JP. Trans-plantation of dermal fibroblasts expressing MyoD1 in mouse muscles. Biochem Biophys Res Commun. 1998;248:648-54.

13. Muschler GF, Boehm C, Easley K. Aspiration to obtain osteoblast progenitor cells from human bone marrow: the influence of aspiration volume. J Bone Joint Surg Am. 1997;79:1699-709. Erratum in: J Bone Joint Surg Am. 1998;80:302.

14. ODriscoll SW. Articular cartilage regeneration using periosteum. Clin Orthop Relat Res. 1999;367 Suppl:S186-203.

15. Owen M, Friedenstein AJ. Stromal stem cells: marrow-derived osteogenic pre-cursors. Ciba Found Symp. 1988;136:42-60.

16. Peng H, Huard J. Muscle-derived stem cells for musculoskeletal tissue regen-eration and repair. Transpl Immunol. 2004;12:311-9.

17. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, Moor-man MA, Simonetti DW, Craig S, Marshak DR. Multilineage potential of adult hu-man mesenchymal stem cells. Science. 1999;284:143-7.

18. Shi S, Gronthos S. Perivascular niche of postnatal mesenchymal stem cells in human bone marrow and dental pulp. J Bone Miner Res. 2003;18:696-704.

19. Bahrami S, Stratmann U, Wiesmann HP, Mokrys K, Bruckner P, Szuwart T. Peri-osteally derived osteoblast-like cells differentiate into chondrocytes in suspen-sion culture in agarose. Anat Rec. 2000;259:124-30.

20. Bosch P, Musgrave D, Ghivizzani S, Latterman C, Day CS, Huard J. The effi-ciency of muscle-derived cell-mediated bone formation. Cell Transplant. 2000;9:463-70.

21. Bosch P, Musgrave DS, Lee JY, Cummins J, Shuler T, Ghivizzani TC, Evans T, Robbins TD, Huard J. Osteoprogenitor cells within skeletal muscle. J Orthop Res. 2000;18:933-44.

22. Bradham DM, Horton WE Jr. In vivo cartilage formation from growth factor


117



modulated articular chondrocytes. Clin Orthop Relat Res. 1998;352:239-49.

23. Caterson EJ, Nesti LJ, Albert T, Danielson K, Tuan R. Application of mesenchy-mal stem cells in the regeneration of musculoskeletal tissues. MedGenMed. 2001;E1.

24. Halvorsen YC, Wilkison WO, Gimble JM. Adipose-derived stromal cellstheir utility and potential in bone formation. Int J Obes Relat Metab Disord. 2000;24 Suppl 4:S41-4.

25. Majors AK, Boehm CA, Nitto H, Midura RJ, Muschler GF. Characterization of human bone marrow stromal cells with respect to osteoblastic differentiation. J Orthop Res. 1997;15:546-57.

26. Zuk PA, Zhu M, Mizuno H, Huang J, Futrell JW, Katz AJ, Benhaim P, Lorenz HP, Hedrick MH. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001;7:211-28.

27. Aubin JE. Advances in the osteoblast lineage. Biochem Cell Biol. 1998;76:899-910.

28. Aubin JE. Osteoprogenitor cell frequency in rat bone marrow stromal popula-tions: role for heterotypic cell-cell interactions in osteoblast differentiation. J Cell Biochem. 1999;72:396-410.

29. Muschler GF, Nitto H, Boehm CA, Easley KA. Age- and gender-related changes in the cellularity of human bone marrow and the prevalence of osteoblastic pro-genitors. J Orthop Res. 2001;19:117-25.

30. Caplan AI. Mesenchymal stem cells. J Orthop Res. 1991;9:641-50.

31. Jiang Y, Jahagirdar BN, Reinhardt RL, Schwartz RE, Keene CD, Ortiz-Gonzalez XR, Reyes M, Lenvik T, Lund T, Blackstad M, Du J, Aldrich S, Lisberg A, Low WC, Largaespada DA, Verfaillie CM. Pluripotency of mesenchymal stem cells derived from adult marrow. Nature. 2002;418:41-9. Erratum in: Nature. 2007;447:879-80.

32. Reyes M, Verfaillie CM. Characterization of multipotent adult progenitor cells, a subpopulation of mesenchymal stem cells. Ann N Y Acad Sci. 2001;938:231-5.

33. Caplan AI. Review: mesenchymal stem cells: cell-based reconstructive ther-apy in orthopedics. Tissue Eng. 2005;11:1198-211.

34. DIppolito G, Schiller PC, Ricordi C, Roos BA, Howard GA. Age-related osteo-genic potential of mesenchymal stromal stem cells from human vertebral bone marrow. J Bone Miner Res. 1999;14:1115-22.

35. Bruder SP, Fox BS. Tissue engineering of bone. Cell based strategies. Clin Or-thop Relat Res. 1999;367 Suppl:S68-83.

36. Pittenger MF, Mosca JD, McIntosh KR. Human mesenchymal stem cells: pro-genitor cells for cartilage, bone, fat and stroma. Curr Top Microbiol Immunol. 2000;251:3-11.

37. Tallheden T, Dennis JE, Lennon DP, Sjgren-Jansson E, Caplan AI, Lindahl A. Phenotypic plasticity of human articular chondrocytes. J Bone Joint Surg Am. 2003;85 Suppl 2:93-100.

38. De Ugarte DA, Morizono K, Elbarbary A, Alfonso Z, Zuk PA, Zhu M, Dragoo JL, Ashjian P, Thomas B, Benhaim P, Chen I, Fraser J, Hedrick MH. Comparison of multi-lineage cells from human adipose tissue and bone marrow. Cells Tissues Organs. 2003;174:101-9.

39. Winter A, Breit S, Parsch D, Benz K, Steck E, Hauner H, Weber RM, Ewerbeck V, Richter W. Cartilage-like gene expression in differentiated human stem cell spheroids: a comparison of bone marrow-derived and adipose tissue-derived stro-mal cells. Arthritis Rheum. 2003;48:418-29.

40. Gronthos S, Franklin DM, Leddy HA, Robey PG, Storms RW, Gimble JM. Sur-face protein characterization of human adipose tissue-derived stromal cells. J Cell Physiol. 2001;189:54-63.

41. Carter DR, Beaupr GS, Giori NJ, Helms JA. Mechanobiology of skeletal re-generation. Clin Orthop Relat Res. 1998;355 Suppl:S41-55.

42. Klein-Nulend J, Roelofsen J, Sterck JG, Semeins CM, Burger EH. Mechanical loading stimulates the release of transforming growth factor-beta activity by cul-tured mouse calvariae and periosteal cells. J Cell Physiol. 1995;163:115-9.

43. Turner CH, Owan I, Alvey T, Hulman J, Hock JM. Recruitment and proliferative responses of osteoblasts after mechanical loading in vivo determined using sustained-release bromodeoxyuridine. Bone. 1998;22:463-9.

44. Aaron RK, Ciombor DM. Acceleration of experimental endochondral ossifica-tion by biophysical stimulation of the progenitor cell pool. J Orthop Res. 1996;14:582-9.

45. Brighton CT, Wang W, Seldes R, Zhang G, Pollack SR. Signal transduction in electrically stimulated bone cells. J Bone Joint Surg Am. 2001;83:1514-23.

46. Patterson TE, Sakai Y, Grabiner MD, Ibiwoye M, Midura RJ, Zborowski M, Wolfman A. Exposure of murine cells to pulsed electromagnetic fields rapidly

activates the mTOR signaling pathway. Bioelectromagnetics. 2006;27:535-44.

47. Rubin C, Bolander M, Ryaby JP, Hadjiargyrou M. The use of low-intensity ultra-sound to accelerate the healing of fractures. J Bone Joint Surg Am. 2001;83:259-70.

48. Cosman F, Nieves J, Woelfert L, Formica C, Gordon S, Shen V, Lindsay R. Par-athyroid hormone added to established hormone therapy: effects on vertebral fracture and maintenance of bone mass after parathyroid hormone withdrawal. J Bone Miner Res. 2001;16:925-31.

49. Finkelstein JS, Klibanski A, Arnold AL, Toth TL, Hornstein MD, Neer RM. Prevention of estrogen deficiency-related bone loss with human parathyroid hormone-(1-34): a randomized controlled trial. JAMA. 1998;280:1067-73.

50. Lane NE, Sanchez S, Modin GW, Genant HK, Pierini E, Arnaud CD. Parathyroid hormone treatment can reverse corticosteroid-induced osteoporosis. Results of a randomized controlled clinical trial. J Clin Invest. 1998;102:1627-33.

51. Morley P, Whitfield JF, Willick GE. Parathyroid hormone: an anabolic treatment for osteoporosis. Curr Pharm Des. 2001;7:671-87.

52. Reeve J, Meunier PJ, Parsons JA, Bernat M, Bijvoet OL, Courpron P, Edouard C, Klenerman L, Neer RM, Renier JC, Slovik D, Vismans FJ, Potts JT Jr. Anabolic effect of human parathyroid hormone fragment on trabecular bone in involutional osteoporosis: a multicentre trial. Br Med J. 1980;280:1340-4.

53. Slovik DM, Rosenthal DI, Doppelt SH, Potts JT Jr, Daly MA, Campbell JA, Neer RM. Restoration of spinal bone in osteoporotic men by treatment with human par-athyroid hormone (1-34) and 1,25-dihydroxyvitamin D. J Bone Miner Res. 1986;1:377-81.

54. Hennessey JV, Chromiak JA, DellaVentura S, Reinert SE, Puhl J, Kiel DP, Rosen CJ, Vandenburgh H, MacLean DB. Growth hormone administration and ex-ercise effects on muscle fiber type and diameter in moderately frail older people. J Am Geriatr Soc. 2001;49:852-8.

55. Liu PY, Wishart SM, Handelsman DJ. A double-blind, placebo-controlled, ran-domized clinical trial of recombinant human chorionic gonadotropin on muscle strength and physical function and activity in older men with partial age-related androgen deficiency. J Clin Endocrinol Metab. 2002;87:3125-35.

56. Khosla S, Eghbali-Fatourechi GZ. Circulating cells with osteogenic potential. Ann N Y Acad Sci. 2006;1068:489-97.

57. Kumagai K, Vasanji A, Drazba JA, Butler RS, Muschler GF. Circulating cells with osteogenic potential are physiologically mobilized into the fracture healing site in the parabiotic mice model. J Orthop Res. 2007 Aug 29 [Epub ahead of print].

58. Muschler GF, Nitto H, Matsukura Y, Boehm C, Valdevit A, Kambic H, Davros W, Powell K, Easley K. Spine fusion using cell matrix composites enriched in bone marrow-derived cells. Clin Orthop Relat Res. 2003;407:102-18.

59. Burwell RG. The fate of bone grafts. In: Apley AG, editor. Recent advances in orthopaedics. London: Churchill; 1969. p 115-207.

60. Burwell RG. Studies in the transplantation of bone. 8. Treated composite homograft-autografts of cancellous bone: an analysis of inductive mechanisms in bone transplantation. J Bone Joint Surg Br. 1966;48:532-66.

61. Burwell RG. The function of bone marrow in the incorporation of a bone graft. Clin Orthop Relat Res. 1985;200:125-41.

62. Connolly JF, Guse R, Tiedeman J, Dehne R. Autologous marrow injection as a substitute for operative grafting of tibial nonunions. Clin Orthop Relat Res. 1991;266:259-70.

63. Connolly JF, Guse R, Tiedeman J, Dehne R. Autologous marrow injection for delayed unions of the tibia: a preliminary report. J Orthop Trauma. 1989;3:276-82.

64. Garg NK, Gaur S, Sharma S. Percutaneous autogenous bone marrow grafting in 20 cases of ununited fracture. Acta Orthop Scand. 1993;64:671-2.

65. Healey JH, Zimmerman PA, McDonnell JM, Lane JM. Percutaneous bone mar-row grafting of delayed union and nonunion in cancer patients. Clin Orthop Relat Res. 1990;256:280-5.

66. Connolly JF. Clinical use of marrow osteoprogenitor cells to stimulate osteo-genesis. Clin Orthop Relat Res. 1998;355 Suppl:S257-66.

67. Connolly JF. Injectable bone marrow preparations to stimulate osteogenic re-pair. Clin Orthop Relat Res. 1995;313:8-18.

68. Hernigou P, Poignard A, Beaujean F, Rouard H. Percutaneous autologous bone-marrow grafting for nonunions. Influence of the number and concentration of progenitor cells. J Bone Joint Surg Am. 2005;87:1430-7.

69. Bittira B, Kuang JQ, Al-Khaldi A, Shum-Tim D, Chiu RC. In vitro preprogram-


118



ming of marrow stromal cells for myocardial regeneration. Ann Thorac Surg. 2002;74:1154-60.

70. Glimm H, Eaves CJ. Direct evidence for multiple self-renewal divisions of hu-man in vivo repopulating hematopoietic cells in short-term culture. Blood. 1999;94:2161-8.

71. Kadiyala S, Young RG, Thiede MA, Bruder SP. Culture expanded canine mes-enchymal stem cells possess osteochondrogenic potential in vivo and in vitro. Cell Transplant. 1997;6:125-34.

72. McKay R. Stem cellshype and hope. Nature. 2000;406:361-4.

73. McNiece I, Briddell R. Ex vivo expansion of hematopoietic progenitor cells and mature cells. Exp Hematol. 2001;29:3-11.

74. Quarto R, Mastrogiacomo M, Cancedda R, Kutepov SM, Mukhachev V, Lavroukov A, Kon E, Marcacci M. Repair of large bone defects with the use of au-tologous bone marrow stromal cells. N Engl J Med. 2001;344:385-6.

75. Ringe J, Kaps C, Burmester GR, Sittinger M. Stem cells for regenerative med-icine: advances in the engineering of tissues and organs. Naturwissenschaften. 2002;89:338-51.

76. Hayflick L, Moorhead PS. The serial cultivation of human diploid cell strains. Exp Cell Res. 1961;25:585-621.

77. Shi S, Gronthos S, Chen S, Reddi A, Counter CM, Robey PG, Wang CY. Bone formation by human postnatal bone marrow stromal stem cells is enhanced by te-lomerase expression. Nat Biotechnol. 2002;20:587-91.

78. Simonsen JL, Rosada C, Serakinci N, Justesen J, Stenderup K, Rattan SI, Jensen TG, Kassem M. Telomerase expression extends the proliferative life-span and maintains the osteogenic potential of human bone marrow stromal cells. Nat Biotechnol. 2002;20:592-6.

79. Bentley G, Biant LC, Carrington RW, Akmal M, Goldberg A, Williams AM, Skin-ner JA, Pringle J. A prospective, randomised comparison of autologous chondro-cyte implantation versus mosaicplasty for osteochondral defects in the knee. J Bone Joint Surg Br. 2003;85:223-30.

80. Horas U, Pelinkovic D, Herr G, Aigner T, Schnettler R. Autologous chondrocyte implantation and osteochondral cylinder transplantation in cartilage repair of the knee joint. A prospective, comparative trial. J Bone Joint Surg Am. 2003;85:185-92.

81. Knutsen G, Engebretsen L, Ludvigsen TC, Drogset JO, Grntvedt T, Solheim E, Strand T, Roberts S, Isaksen V, Johansen O. Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. J Bone Joint Surg Am. 2004;86:455-64.

82. Hannallah D, Peterson B, Lieberman JR, Fu FH, Huard J. Gene therapy in or-thopaedic surgery. Instr Course Lect. 2003;52:753-68.

83. Bonadio J. Tissue engineering via local gene delivery. J Mol Med. 2000;78:303-11.

84. Prockop DJ. Marrow stromal cells as stem cells for nonhematopoietic tis-sues. Science. 1997;276:71-4.

85. Hunziker EB. Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects. Osteoarthritis Cartilage. 2002;10:432-63.

86. Peterson L. Articular cartilage injuries treated with autologous chondrocyte transplantation in the human knee. Acta Orthop Belg. 1996;62 Suppl 1:196-200.

87. Choueka J, Charvet JL, Koval KJ, Alexander H, James KS, Hooper KA, Kohn J. Canine bone response to tyrosine-derived polycarbonates and poly(L-lactic acid). J Biomed Mater Res. 1996;31:35-41.

88. Ertel SI, Kohn J, Zimmerman MC, Parsons JR. Evaluation of poly(DTH carbon-ate), a tyrosine-derived degradable polymer, for orthopedic applications. J Biomed Mater Res. 1995;29:1337-48.

89. Meechaisue C, Dubin R, Supaphol P, Hoven VP, Kohn J. Electrospun mat of tyrosine-derived polycarbonate fibers for potential use as tissue scaffolding mate-rial. J Biomater Sci Polym Ed. 2006;17:1039-56.

90. Payne RG, McGonigle JS, Yaszemski MJ, Yasko AW, Mikos AG. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 2. Viability of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate). Biomaterials. 2002;23:4373-80.

91. Payne RG, McGonigle JS, Yaszemski MJ, Yasko AW, Mikos AG. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell populations. Part 3. Proliferation and differentiation of encapsulated marrow stromal osteoblasts cultured on crosslinking poly(propylene fumarate). Biomateri-als. 2002;23:4381-7.

92. Payne RG, Yaszemski MJ, Yasko AW, Mikos AG. Development of an injectable, in situ crosslinkable, degradable polymeric carrier for osteogenic cell popula-tions. Part 1. Encapsulation of marrow stromal osteoblasts in surface crosslinked gelatin microparticles. Biomaterials. 2002;23:4359-71.

93. Wang S, Lu L, Yaszemski MJ. Bone-tissue-engineering material poly(propylene fumarate): correlation between molecular weight, chain dimensions, and physical properties. Biomacromolecules. 2006;7:1976-82.

94. Uludag H, DAugusta D, Golden J, Li J, Timony G, Reidel R, Wozney JM. Im-plantation of human recombinant bone morphogenetic proteins with biomaterial carriers: a correlation between protein pharmacokinetics and osteoinduction in the rat ectopic model. J Biomed Mater Res. 2000;50:227-38.

95. Boden SD, Kang J, Sandhu H, Heller JG. Use of recombinant human bone morphogenetic protein-2 to achieve posterolateral lumbar spine fusion in hu-mans: a prospective, randomized clinical pilot trial. Spine. 2002;27:2662-73.

96. Griffith LG. Polymeric biomaterials. Acta Mater. 2000;48:263-77.

97. Altankov G, Thom V, Groth T, Jankova K, Jonsson G, Ulbricht M. Modulating the biocompatibility of polymer surfaces with poly(ethylene glycol): effect of fi-bronectin. J Biomed Mater Res. 2000;52:219-30.

98. Groth T, Altankov G. Fibroblast spreading and proliferation on hydrophilic and hydrophobic surfaces is related to tyrosine phosphorylation in focal contacts. J Biomater Sci Polym Ed. 1995;7:297-305.

99. Groth T, Altankov G, Kostadinova A, Krasteva N, Albrecht W, Paul D. Altered vitronectin receptor (alphav integrin) function in fibroblasts adhering on hydropho-bic glass. J Biomed Mater Res. 1999;44:341-51.

100. Lewandowska K, Balachander N, Sukenik CN, Culp LA. Modulation of fi-bronectin adhesive functions for fibroblasts and neural cells by chemically deriva-tized substrata. J Cell Physiol. 1989;141:334-45.

101. Burkus JK, Heim SE, Gornet MF, Zdeblick TA. Is INFUSE bone graft superior to autograft bone? An integrated analysis of clinical trials using the LT-CAGE lum-bar tapered fusion device. J Spinal Disord Tech. 2003;16:113-22.

102. Burkus JK, Transfeldt EE, Kitchel SH, Watkins RG, Balderston RA. Clinical and radiographic outcomes of anterior lumbar interbody fusion using recombinant human bone morphogenetic protein-2. Spine. 2002;27:2396-408.

103. Dennis JE, Caplan AI. Porous ceramic vehicles for rat-marrow-derived (Rat-tus norvegicus) osteogenic cell delivery: effects of pre-treatment with fibronectin or laminin. J Oral Implantol. 1993;19:106-15,136-7.

104. Dennis JE, Haynesworth SE, Young RG, Caplan AI. Osteogenesis in marrow-derived mesenchymal cell porous ceramic composites transplanted subcutane-ously: effect of fibronectin and laminin on cell retention and rate of osteogenic expression. Cell Transplant. 1992;1:23-32.

105. Johnsson R, Strmqvist B, Aspenberg P. Randomized radiostereometric study comparing osteogenic protein-1 (BMP-7) and autograft bone in human non-instrumented posterolateral lumbar fusion. Spine. 2002;27:2654-61.

106. Griffith LG. Emerging design principles in biomaterials and scaffolds for tis-sue engineering. Ann N Y Acad Sci. 2002;961:83-95.

107. Griffith LG, Naughton GK. Tissue engineeringcurrent challenges and ex-panding opportunities. Science. 2002;295:1009-14.

108. Kuhl PR, Griffith-Cima LG. Tethered epidermal growth factor as a paradigm for growth factor-induced stimulation from the solid phase. Nat Med. 1996;2:1022-27. Erratum in: Nat Med. 1997;3:93.

109. Mann BK, Schmedlen RH, West JL. Tethered-TGF-beta increases extracellu-lar matrix production of vascular smooth muscle cells. Biomaterials. 2001;22:439-44.

110. Swindle CS, Tran KT, Johnson TD, Banerjee P, Mayes AM, Griffith L, Wells A. Epidermal growth factor (EGF)-like repeats of human tenascin-C as ligands for EGF receptor. J Cell Biol. 2001;154:459-68.

111. Zisch AH, Schenk U, Schense JC, Sakiyama-Elbert SE, Hubbell JA. Co-valently conjugated VEGFfibrin matrices for endothelialization. J Contr Rel. 2001;72:101-13.

112. Fischgrund JS, James SB, Chabot MC, Hankin R, Herkowitz HN, Wozney JM, Shirkhoda A. Augmentation of autograft using rhBMP-2 and different carrier me-dia in the canine spinal fusion model. J Spinal Disord. 1997;10:467-72.

113. Isobe M, Yamazaki Y, Mori M, Amagasa T. Bone regeneration produced in rat femur defects by polymer capsules containing recombinant human bone morpho-genetic protein-2. J Oral Maxillofac Surg. 1999;57:695-9.

114. Isobe M, Yamazaki Y, Mori M, Ishihara K, Nakabayashi N, Amagasa T. The role of recombinant human bone morphogenetic protein-2 in PLGA capsules at an extraskeletal site of the rat. J Biomed Mater Res. 1999;45:36-41.


119



115. Kandziora F, Bail H, Schmidmaier G, Schollmeier G, Scholz M, Knispel C, Hiller T, Pflugmacher R, Mittlmeier T, Raschke M, Haas NP. Bone morphogenetic protein-2 application by a poly(D,L-lactide)-coated interbody cage: in vivo results of a new carrier for growth factors. J Neurosurg. 2002;97(1 Suppl):40-8.

116. Kenley R, Marden L, Turek T, Jin L, Ron E, Hollinger JO. Osseous regenera-tion in the rat calvarium using novel delivery systems for recombinant human bone morphogenetic protein-2 (rhBMP-2). J Biomed Mater Res. 1994;28:1139-47.

117. Mayer M, Hollinger J, Ron E, Wozney J. Maxillary alveolar cleft repair in dogs using recombinant human bone morphogenetic protein-2 and a polymer carrier. Plast Reconstr Surg. 1996;98:247-59.

118. Weber FE, Eyrich G, Grtz KW, Maly FE, Sailer HF. Slow and continuous appli-cation of human recombinant bone morphogenetic protein via biodegradable poly(lactide-co-glycolide) foamspheres. Int J Oral Maxillofac Surg. 2002;31:60-5.

119. Woo BH, Fink BF, Page R, Schrier JA, Jo YW, Jiang G, DeLuca M, Vasconez HC, DeLuca PP. Enhancement of bone growth by sustained delivery of recombi-nant human bone morphogenetic protein-2 in a polymeric matrix. Pharm Res. 2001;18:1747-53. Erratum in: Pharm Res. 2003;20:334.

120. Hecht BP, Fischgrund JS, Herkowitz HN, Penman L, Toth JM, Shirkhoda A.

The use of recombinant human bone morphogenetic protein 2 (rhBMP-2) to pro-mote spinal fusion in a nonhuman primate anterior interbody fusion model. Spine. 1999;24:629-36.

121. Hollinger JO, Schmitt JM, Buck DC, Shannon R, Joh SP, Zegzula HD, Wozney J. Recombinant human bone morphogenetic protein-2 and collagen for bone re-generation. J Biomed Mater Res. 1998;43:356-64.

122. Marden LJ, Hollinger JO, Chaudhari A, Turek T, Schaub RG, Ron E. Recombi-nant human bone morphogenetic protein-2 is superior to demineralized bone ma-trix in repairing craniotomy defects in rats. J Biomed Mater Res. 1994;28:1127-38.

123. Magin MN, Delling G. Improved lumbar vertebral interbody fusion using rhOP-1: a comparison of autogenous bone graft, bovine hydroxylapatite (Bio-Oss), and BMP-7 (rhOP-1) in sheep. Spine. 2001;26:469-78.

124. Ripamonti U, Ramoshebi LN, Matsaba T, Tasker J, Crooks J, Teare J. Bone induction by BMPs/OPs and related family members in primates. J Bone Joint Surg Am. 2001;83 Suppl 1:S116-27.

125. Brekke JH, Toth JM. Principles of tissue engineering applied to programma-ble osteogenesis. J Biomed Mater Res. 1998;43:380-98. Erratum in: J Biomed Mater Res. 1999;48:95.


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Cellular Strategies for Fracture Repair