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Osteoinduction : A Review of Old Concepts with New Standards R.J. Miron and Y.F. Zhang J DENT RES 2012 91: 736 originally published online 8 February 2012 DOI: 10.1177/0022034511435260 The online version of this article can be found at: http://jdr.sagepub.com/content/91/8/736

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CRITICAL REVIEWS IN ORAL BIOLOGY & MEDICINE R.J. Miron1,2* and Y.F. Zhang1* 1

The State Key Laboratory Breeding Base of Basic Science of Stomatology (Hubei-MOST) & Key Laboratory of Oral Biomedicine, Ministry of Education, School & Hospital of Stomatology, Wuhan University, 237 LuoYu Road, Wuhan, 430079, China; and 2Faculté de médecine dentaire, Pavillon de médecine dentaire, rue de la Terrasse, Université Laval, Québec, Canada; *corresponding authors, richard. [email protected] and [email protected]

Osteoinduction: A Review of Old Concepts with New Standards

J Dent Res 91(8):736-744, 2012

Abstract Since the discovery of osteoinduction in the early 20th century, innovative biomaterials with osteoinductive potential have emerged. Over the last 50 years, however, our ability to describe biological phenomena accurately has been improved dramatically by advancements in cell and molecular biology. The aim of this review is to divide the osteoinduction phenomenon into 3 principles: (1) mesenchymal cell recruitment, (2) mesenchymal differentiation to bone-forming osteoblasts, and (3) ectopic bone formation in vivo. Furthermore, this review formulates guidelines for in vitro and in vivo experimental testing for accurately defining new biomaterials as osteoinductive. The use of growth factors with osteoinductive potential in periodontal and oral surgery is discussed. These concepts and guidelines aim to guide the future direction of emerging biomaterials in bone regeneration.

KEY WORDS: PDGF, BMP, bone regeneration, bone remodeling, osteoinduction, bone grafts.

Overview

F

rom the discovery of bone morphogenic proteins (Urist and Strates, 1971) to the advancements in tissue engineering (Langer, 2009), the field of bone regeneration has tackled a wide variety of surgical issues caused by trauma, bone loss diseases, infections, biochemical disorders, and abnormal skeletal development. One such discipline which applies the principles of biology to the development of viable substitutes is the domain of biomaterials, which is an emerging interdisciplinary field aimed to restore, maintain, or improve the function of human tissues (Anderson et al., 2004). Over the years, the role of biomaterials has changed from a passive, structural supporting network to one that will orchestrate the process of tissue engineering. These are best exemplified in bone-grafting procedures, more than 2.2 million of which are performed yearly at an estimated global cost approaching $2.5 billion annually (Giannoudis et al., 2005). As the population ages, the number of bone-grafting procedures for diseases such as osteoporosis, arthritis, tumors, and trauma also increases, placing an even larger demand on the healthcare system to replace and restore lost bone. With markets increasing by over 50% annually and sales of regenerative biomaterials exceeding US$240 million per annum (Place et al., 2009), improved understanding of fundamental concepts is necessary. The regenerative potential of bone grafts is governed by 3 fundamental mechanisms. The ideal grafting material should provide: (1) an osteoconductive matrix, which allows for vascular invasion and cellular infiltration; (2) osteoinductive factors, which recruit and induce mesenchymal cells to differentiate into mature bone-forming cells; and (3) osteogenic cells, contained inside the bone graft, capable of laying new bone matrix.

DOI: 10.1177/0022034511435260 Received September 19, 2011; Last revision December 11, 2011; Accepted December 13, 2011 A supplemental appendix to this article is published electronically only at http://jdr.sagepub.com/supplemental. © International & American Associations for Dental Research

Consequently, the gold standard for bone-grafting procedures is autogenous bone because of its excellent combination of osteoconduction and osteoinduction. Despite their benefits, limitations of additional surgical procedures and donor site morbidity have necessitated the pursuit of alternatives. These include freeze-dried bone allografts from human donors, xenografts from animal models, and an array of synthetic alloplasts such as hydroxyapatite, βTCP, polymers, and bioactive glass (Kao and Scott, 2007). Other strategies incorporate growth-factor- and cell-based alternatives used either alone or in combination with other materials (Jabbarzadeh et al., 2008). Although most

736 Downloaded from jdr.sagepub.com at UNIVERSITE LAVAL on November 17, 2012 For personal use only. No other uses without permission. © 2012 International & American Associations for Dental Research

J Dent Res 91(8) 2012

Osteoinduction: Old Concepts, New Standards 737

Table 1. The Osteoinductive Bone Grafts Approved by the US FDA and the EU Marketing Year

Product Name

2000

DBX

2001

Collagraft

2001

Tutoplast®

2002

INFUSE® Bone Graft

2002

DynaGraft®

2004

InductOs®

2004

Allomatrix

2005

OrthoBlast®~II

2005

Grafton

2005

Osteofil®

2005

InterGro®DBM

2007

NuBone™

2008

OraGRAFT®

2009

Pro-Stim Injectable Inductive Graft

2009

EquivaBone®

2010

OP-1™

2011

Accell TBM (R)

Composition

Clinical Phase

Demineralized bone matrix (DBM) in sodium hyaluronate carrier 65% hydroxyapatite, 35% tricalcium phosphate and bovine collagen Allograft bone Bone morphogenetic protein-2 (BMP-2) in absorbable collagen sponge Demineralized bone matrix (DBM) mixed with temp. sensitive polymer Human bone morphogenetic protein-2 (BMP-2) in bovine collagen sponge carrier Demineralized bone matrix (DBM) and cancellous bone chips with calcium sulfate carrier Demineralized bone matrix (DBM)

Approved by

On market

MTF (Edison, NJ, USA)

US FDA

On market

Neucoll (Campbell, CA, USA)

US FDA and EU

On market

Tutogen Medicals (Clifton, NJ,USA) Medtronic (Minneapolis, MN, USA) IsoTis (Irvine, CA,USA)

US FDA

On market

On market

On market

US FDA

US FDA

Wyeth Pharmaceuticals (NewLane Havant, Hants, UK) Wright Medical (Arlington, TN, USA)

US FDA and EU

lsoTis OrthoBiologics, Inc. (Irvine, CA, USA) Osteotech (Eatontown, NJ, USA) Regeneration Technology (Alachua, FL, USA) Interpore Cross International (Irvine, CA, USA) Globus Medical, Inc. (Audubon, PA, USA)

US FDA and EU

LifeNet Health, Inc. (Virginia Beach, VA, USA) Wright Medical Group (Arlington, TN, USA)

US FDA

On market

ETEX Corporation (Cambridge, MA, USA)

US FDA

On market

Stryker (Kalamazoo, MI, USA) Integra LifeSciences Holdings Corporation (Plainsboro, NJ, USA)

US FDA and EU

On market

On market

Demineralized bone matrix (DBM) fibers with glycerol carrier Demineralized bone matrix (DBM) particulate with porcine collagen carrier Demineralized allograft bone in lipid carrier

On market

Allograft tissue (demineralized bone matrix [(DBM)] combined with cortical bone gelatin carrier) Demineralized Freeze-dried Bone Allograft

On market

Calcium sulfate and calcium phosphate materials with osteoinductive demineralized bone matrix Osteoinductive Demineralized Bone Matrix (DBM) and osteoconductive nanocrystalline calcium phosphate Combination of Osteogenic Protein 1 and a collagen carrier Demineralized bone matrix (DBM)

On market

of these materials are osteoconductive to bone-forming osteoblasts, only a limited number of osteoinductive materials are currently available on the market with FDA approval (Table 1). Many investigators have aimed to generate new biomaterials with osteoinductive potential; however, a fundamental understanding of the term is of critical importance. The aim of this review is to clarify the original definition as presented by Marshall Urist in 1965, to generate new insights with the aid of advancements in cell biology over the last 50 years, and to list

Company

On market

On market

On market

On market

US FDA and EU

US FDA and EU US FDA

US FDA

US FDA

US FDA

US FDA

necessary in vitro and in vivo experiments for the accurate definition of new biomaterials as osteoinductive.

Osteoinduction as originally defined Historically, osteoinduction refers to the process by which one tissue, or product derived from it, causes a second undifferentiated tissue to differentiate into bone. The earliest proof of bone induction was observed in autoimplantation of transitional epithelium of the


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showed more osteogenic activity than DBM alone. The original definition of osteoinduction was defined as “the mechanism of cellular differentiation toward bone of one tissue due to physicochemical effect or contact with another tissue” (Urist et al., 1967).

The ideal osteoinductive material To maximize the potential of osteoinductive biomaterials, the osteoinduction phenomenon is divided into 3 individual mechanisms/principles (Fig. 1). As defined by Urist, an osteoinductive material should first be capable of recruiting mesenchymal-type osteoprogenitor cells. Investigators’ ability to study migration patterns, growth factor and cytokine release, as well as behavioral cell analysis has drastically improved over the last 40 years and will be addressed as Principle One. Second, the ideal osteoinductive material should be capable of transforming an undifferentiated mesenchymal cell into a mature, bone-forming osteoblast. For this to occur, an understanding of mesenchymal differentiation to cartilage and bone is summarized as Principle Two. Last, the ideal osteoinductive material should be capable of inducing in-growth ectopic bone formation when implanted into extraskeletal locations. This phenomenon will be addressed as Principle Three. The future design of new osteoinductive biomaterials aimed to maximize these components will ultimately generate the next generation of innovative biomaterials.

Osteoinductive principle 1: MSC recruitment

Figure 1. Principles of osteoinductive materials. Principle 1: Osteoinductive materials should be capable of recruiting MSCs to bone graft surfaces through growth factor release. Principle 2: The material should promote MSC differentiation into osteoblasts. Principle 3: Osteoblasts must be capable of forming ectopic bone in vivo.

urinary bladder to the abdominal wall muscles in dogs (Huggins, 1931). This discovery was further investigated by Levander (1938), who demonstrated that crude alcoholic extracts of bone induced bone formation when injected into muscle tissue. Over the following 20 years, many authors investigated the osteoinductive phenomenon by studying implantation of bone by methods of demineralization into various body organs to test for an osteopromotive effect. In a classic study by Urist in 1965, he described ‘autoinduction’ by studying the ability of demineralized bone matrix to induce ingrowth of connective tissue and differentiation of cartilage and bone when implanted into extraskeletal locations in rabbits, dogs, and rats. Subsequent investigations from Urist and Reddi demonstrated that low-molecular-weight proteins extracted from demineralized bone matrix (DBM), termed bone morphogenetic proteins (BMPs),

Over 40 years ago, Friedenstein first reported evidence of multipotential mesenchymal cells (MSCs) that were isolated from bone marrow and formed fibroblast-like colonies with multipotential to differentiate into adipocytes, chondrocytes, osteoblasts, and myoblasts (Friedenstein et al., 1970). Since then, research focused on stem cells has gathered tremendous momentum. MSCs have now been identified not only in mesodermal tissues (bone marrow, trabecular bone, synovium, cartilage, fat, muscle, and tonsil) but also in endoderm (thymus, liver)- and ectoderm (skin, hair follicle, dura mater, and dental pulp)derived tissues (Phinney and Prockop, 2007). They are a heterogeneous population of pluripotent progenitor cells that are now capable of differentiating into osteoblasts, chondrocytes, adipocytes, myocytes,cardiomyocytes, fibroblasts, myofibroblasts, epithelial cells, and neurons (Liu et al., 2009). Currently, MSCs are defined by an array of phenotypic markers and ability for multipotential differentiation, as defined in a position paper (Horwitz et al., 2005). The International Society for Cellular Therapy has provided the following minimal criteria for defining multipotential MSCs: (1) plastic-adherent under standard culture conditions; (2) positive for CD105, CD73, and CD90 and negative for CD34, CD45, CD11a, CD19, and HLA-DR; and (3) under standard differentiation conditions, MSCs should differentiate into osteocytes, adipocytes, and chondrocytes in vitro. Due to a growing interest in MSC cell-based therapy (De Bari and Dell’accio, 2008), a plethora of research and clinical


J Dent Res 91(8) 2012


trials has generated enormous advances in our current understanding. Initially, MSCs were tested in cancer therapy by the addition of expanded human MSCs to bone marrow transplantations, with the assumption that MSCs could rejuvenate the bone marrow stroma of chemotherapy-/radiation-treated patients (Lazarus et al., 1995, 2005). Since then, MSCs have been used to treat heart infarct ischemia, stroke ischemia, meniscus regeneration, tendinitis, spinal cord interruption (contusion or cuts), and repair of damaged cartilage, bone, and muscle (Barry and Murphy, 2004; De Bari and Dell’accio, 2008). It has long been believed that growth factors also have the potential to accelerate healing and tissue regeneration in challenging scenarios by recruiting MSCs to sites of injury (Kaigler et al., 2011). Although the precise molecular mechanisms that govern MSC migration to sites of injury are not yet fully understood (MendezFerrer et al., 2010), the study of leukocyte migration (Heinzel et al., 2007) and fracture healing (Ai-Aql et al., 2008) has provided a paradigm for MSC homing. During fracture healing, several growth factors, including BMPs as well as plateletderived growth factors (PDGFs), have been shown to rapidly stimulate MSC recruitment to sites of injury (Ai-Aql et al., 2008). BMPs are pleiotropic morphogens that induce a sequential cascade of events, including chemotaxis, regulation of growth, differentiation, angiogenesis, and apoptosis (LissenbergThunnissen et al., 2011). In vitro and in vivo studies have demonstrated that BMPs are capable of enhancing osteoinductivity of MSCs and regulate their proliferation and differentiation into osteoblasts, making them well suitable for osteoinductive materials (Lissenberg-Thunnissen et al., 2011). The ability of BMPs to recruit MSCs and osteoblasts has been demonstrated with modified chemotaxis Boyden and Dunn chemotaxis chambers (Li et al., 2005; Gonnerman et al., 2006). Another growth factor capable of rapid recruitment of MSCs is PDGF, which is available in different forms, including PDGF-AA, -AB, and -BB (Alvarez et al., 2006). Once released from platelets, it acts in sites of injury by promoting rapid cell migration and proliferation (Ronnstrand and Heldin, 2001). Many studies have demonstrated its role as a potent chemotactic factor for gingival and PDL fibroblasts, cementoblasts, and osteoblasts (Kaigler et al., 2011). Primary human MSCs were examined for chemotaxis, and results were expressed as a chemotactic index (CI) (Fiedler et al., 2002). The increase of CI was up to 3.5-fold for rhBMP-2, 3.6-fold for rhBMP-4, and up to 22-fold for rhPDGF-bb, demonstrating the ability for PDGF to drastically stimulate recruitment of MSCs to sites of injury.

Osteoinduction principle 2: MSC differentiation to osteoblasts Although the differentiation of bone of mesenchymal origin is a complex and still unresolved topic, the aims of this article are to summarize present literature which supports the differentiation of mature osteoblasts (Nombela-Arrieta et al., 2011). Osteoblasts are derived from mesenchymal stem cells that can also give rise to myoblasts, adipocytes, and chondrocytes (Ducy et al., 2000). Mesenchymal differentiation to osteoblasts

requires the expression of 2 essential transcription factors: Runtrelated transcription factor 2 (Runx2) (Banerjee et al., 1997; Ducy et al., 1997) and osterix (Nakashima et al., 2002). The importance of Runx2 became apparent from the Runx2-null mouse, which had a cartilaginous skeleton and a complete absence of osteoblasts (Banerjee et al., 1997; Ducy et al., 1997). The osteoblasts from these knockout mice were incapable of entering the mineralization cycle required for bone formation (Ducy et al., 1997). Osterix is a zinc-finger-like factor containing protein which is induced by BMPs. Knockout mice develop perfectly patterned skeletons that lack bone and are composed entirely of cartilage. Osterix was determined to act downstream of Runx2; this was discovered through a lack of expression of osterix in the Runx2 knockout mice, while the expression of Runx2 is present in osterix knockout animals (Nakashima et al., 2002). Runx2 is considered by many to be the ‘master gene’ for osteoblast differentiation, because it regulates the differentiation of mesenchymal progenitor cells to pre-osteoblasts and is required for the expression of non-collagenous proteins such as bone sialoprotein (BSP) and osteocalcin (OC) (Fig. 2). These osteoprogenitor cells begin to produce collagen 1, the most abundant collagen in the body, which provides the structural framework by allowing for dispersion of pressure, torsion, and tension resulting from movement and normal activity (Boskey et al., 1999). Osteoprogenitor cells partially differentiate to pre-osteoblasts, which are characterized by their expression of alkaline phosphatase (ALP), an early marker of osteoblast differentiation (Tenenbaum, 1987). In vitro experiments have demonstrated that ALP participates in the initiation of mineralization (Tenenbaum, 1987; Wennberg et al., 2000). When pre-osteoblasts transform to fully differentiated osteoblasts, elevated expression of BSP and OC occurs (Fig. 2). BSP is a non-collagenous protein with a molecular weight of 70-80 kDa and accounts for approximately 5 to 10% of the non-collagenous proteins of the bone extracellular matrix (Oldberg et al., 1986, 1988; Fisher et al., 1990). It preferentially binds to collagen I and nucleates hydroxyapatite crystal formation, indicating a role in the initial mineralization of bone (Tye et al., 2005). OC is the most abundant osteoblast-specific non-collagenous protein, with a molecular weight of 5.7 kDa. It contains a single chain of 46-50 amino acids and is involved with the binding of calcium and hydroxyapatite (Hauschka et al., 1989; Weinreb et al., 1990; Chenu et al., 1994). Both BSP and OC play a crucial role in bone formation and bone turnover. Fully matured osteoblasts are characterized by their ability to synthesize osteoid, the organic phase of the bone matrix. This osteoid becomes mineralized by the formation of hydroxyapatite (Burger and Klein-Nulend, 1999). As an osteoblast becomes surrounded by its own matrix, it terminally differentiates into an osteocyte and plays a central role in cell communication and the regulation of bone remodeling (Burger and Klein-Nulend, 1999). Otherwise, a mature osteoblast becomes a lining cell or undergoes apoptosis. To compare the ability of growth factors to promote an osteogenic phenotype, investigators have utilized and compared MSCs for cell proliferation, ALP activity, and mineralized matrix. Results demonstrate a proliferating effect for BMPs and


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osteoinductive material by its ability to form bone in a non-bone defect where bone would otherwise not grow. The ability for a bone graft to induce new bone formation in an intraosseous defect does not fully reflect its true osteoinductive potential. There are two ways to test osteoinduction in vivo. One is to implant subcutaneously, and the other is to implant into intramuscular defects (Urist and McLean, 1952). For subcutaneous defects, a rabbit study at 4 wks Figure 2. A list of differentiation markers for MSCs, pre-osteoblasts, osteoblasts, and osteocytes with HA-TCP (Catros et al., 2009) and a during MSC differentiation to osteoblasts and osteocytes. rat study at 3 wks with a controlledrelease system for BMP encapsulated PGDF; however, most studies demonstrate the ability of BMPs with a poly (DL-lactide-co-glycolide) (PLGA)/poly-ethylenegto induce osteoblast differentiation when compared with PDGF lycol (PEG) mixture (Isobe, 1995) showed new bone formation. or platelet-rich plasma (Yu et al., 1997; Chaudhary et al., 2004; A comparative study also demonstrated that transplantation of Wildemann et al., 2007; Mooren et al., 2010). Recently, it was osteogenically undifferentiated MSCs with BMP-2 delivery demonstrated that the combined use of PDGF-BB with synthetic results in more extensive bone formation in subcutaneous peptides could result in an increase in proliferation and osteoregions than that of osteogenically differentiated or undifferentiblast markers, including ALP, collagen, and OC (Vordemvenne ated MSCs (Kang et al., 2010). et al., 2011). PDGF activates and modulates the extracellular Ectopic bone formation models, especially in intramuscular signal-regulated kinase (ERK) pathway (Kim et al., 2007; defects, have a much broader range of animal studies, including Getachew et al., 2010), which has previously been shown to those in immunodeficient mice, normal mice, rats, calves, influence proliferation and differentiation of many cell types, chickens, rabbits, cats, dogs, and sheep (Table 2). In immunoincluding MSCs (Zhang and Liu, 2002). Inhibition of PDGF compromised Nu/Nu mouse muscle, human DBM formulated receptor-beta by imatinib mesylate (IM) significantly reduced with hyaluronic acid and cancellous/cortical bone granules were downstream Akt and ERK1/2 phosphorylation with human implanted and formed bone at 8 wks (Braccini et al., 2005). In bone-marrow-derived MSCs in vitro (Fierro et al., 2007). Swiss albino mice, Fibrin glue combined with calcium phosResults demonstrate that proliferation of MSCs was signifiphate and glass ceramic was implanted into muscle and formed cantly reduced by IM and favored adipogenic differentiation bone after 4 wks (Abiraman et al., 2002). In a study that com(Fierro et al., 2007). Specifically, ERK pathways can activate pared 6 bone graft substitutes (Pyrost, natural coral, Callopat, RUNX2 transcription factor and block PPAR-gamma during Surgibone, demineralized Surgibone, and demineralized rat osteoblast differentiation (Xiao et al., 2000; Kim et al., 2001). bone) implanted into the abdominal musculature of rats, there Many investigators have demonstrated the ability of BMP to was new bone formation after 3 and 6 wks in all groups (Begley induce osteoblast differentiation, both alone or in combination with et al., 1995). In sheep studies, 12 post-implantation wks were various other bone-grafting materials (Appendix Table). It is generrequired for new bone formation in osteoinductive putties comally accepted that BMP-signaling pathways are primarily mediated bining the microstructure of biphasic calcium phosphate partithrough Smad proteins (Massagué, 1996; Miyazono et al., 2001); cles with 5 different polymeric gels (Appendix Table). The however, accumulating evidence demonstrates that many other osteoinductive potential of 2 calcium phosphate ceramics was pathways, including MAPK/Erk, are clearly involved in BMP2evaluated after intramuscular implantation in goats, and new induced osteoblast differentiation (Ryoo et al., 2006). One tranbone was formed 12 wks post-implantation (Appendix Table). A scription factor that plays a key role in BMP2-induced osteoblast full list of studies, including animals, sites of injury, lengths of differentiation is Dlx5 (Ryoo et al., 1997). In vitro experiments studies, and materials used, is presented in Table 2 (Barradas have demonstrated that Dlx5 can be stimulated by endogenous et al., 2011; Habibovic and de Groot, 2007). BMP2, by overexpression of active forms of BMP receptors, or by Growth factors most commonly used in periodontal and oral overexpression of Smad1 and Smad5 proteins (Lee et al., 2003). surgery with osteoinductive potential include BMP2 and PDGF Despite a lack of complete understanding of BMP cellular path(Appendix Table). Various combinations of recombinant BMP2 ways, addition of BMPs remains the growth factor of choice to have been injected into rat muscle and formed new bone 9 and induce MSC differentiation to osteoblasts. 21 days post-implantation (Appendix Table). A plasmid encoding human BMP2 (pCAGGS-BMP-2) was injected repeatedly (from 1 to 8 times) into the skeletal muscle of mice for 8 days Osteoinductive principle 3: and displayed new bone formation (Appendix Table). The comEctopic bone formation bination of BMP2 with various biomaterials has also demonIn his original studies on osteoinduction, Marshall Urist strated ectopic bone formation in vivo. BMP-2 loaded TCP/HAP implanted DBM particles into intramuscular defects and found porous ceramics implanted subcutaneously for 6 wks (Appendix new bone formation around these particles. He later defined an Table), rhBMP-2 incorporated into chitosan and hyaluronan Downloaded from jdr.sagepub.com at UNIVERSITE LAVAL on November 17, 2012 For personal use only. No other uses without permission. © 2012 International & American Associations for Dental Research

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Table 2. The in vivo Study to Test Osteoinductive Materials Animal

Site

Bone-forming Time

Materials Types

Female Swiss Webster mice weighing 12-15 g 8- to 10-week-old female C3H/HeN mice Thirty inbred male Nu/Nu mice Twelve-week-old male C57Bl/6 mice Male 8-week-old ICR mice

Muscle

4-6 wks

60% collagen and 40% DFDBA

Subcutaneously and Muscle Muscle Muscle Muscle

2 wks

10- to 12-week-old male Balb/C mice 6- to 8-week-old male syngeneic C57BL/6J mice 9-week-old F 344/N Jcl-rnu nude rat and 9-week-old Wistar rats Rats

Muscle Muscle

3 wks 6 wks

Oxygen carrier-enriched hydrogel combined with mesenchymal stem cell expressed BMP2 Human demineralized, freeze-dried bone graft BMP2 or BMP9 A plasmid encoding human BMP-2 (pCAGGSBMP-2) BMP2 protein in 0.9% saline or gelatine gel BMP2 expression MSCs seeded on AlloDerm

Muscle

3 wks

Subcutaneously

5 wks

Six-week-old Sprague-Dawley female rat Sprague-Dawley rats

Subcutaneously Muscle

6 wks 3 wks

Rats 5- to 7-week-old immunocompetent Sprague-Dawley rats (SD rats) Four-week-old syngeneic Fisher 344 male rats

Subcutaneous Muscle

3, 6, 12 wks 8 wks 3 wks

Nude rat

Subcutaneously and intramuscularly (No difference) Muscle

Rat

Subcutaneous

5 wks

Rabbit Rabbits Adult female sheep

3, 9 wks 5 wks 1 yr

Sheep Sheep Adult Dutch milk goats

Cranial periosteum Muscle Subcutaneous and intramuscular Muscle Muscle Intramuscularly

Goat Goat

Muscle Muscle

12 wk 12 wks

Goat

Muscle

6, 12 wks

Milk goat Goat Dog Dog Baboon Baboon Rabbits/Dogs/Baboons Rabbit/Dog Dog/Pig/Goat/Rabbit/Rats

Muscle Muscle Subcutaneous/Muscle Muscle Muscle Muscle Muscle Subcutaneous/Muscle Subcutaneous/Muscle

Mice/Rats/Rabbit/Dogs

Subcutaneous/Muscle

6, 12 wks 12 wks 4 mos 12 wks 3, 6, 9 mos 90 days 90 days 3, 6 wks/8 wks 15, 30, 45, 60, 90, and 120 days 90 days

8 wks 2 wks 3 wks

4 wks

12 wks 12 wks 6, 12 wks

Human bone morphogenetic protein-2 (BMP-2) -expressing recombinant adenoviral vector Coating deproteinized bovine bone (DBB) with a layer of calcium phosphate into which bone morphogenetic protein 2 (BMP-2) is incorporated RhBMP-2 containing TCP/HAP granules RhBMP-2 was incorporated into chitosan (CH) and hyaluronan (HY) hydrogels Porous calcium phosphate ceramics Adeno-associated virus (AAV) carrying bone morphogenetic protein2 6-day osteogenic medium differentiation rat bone marrow cells combined with titanium-fiber mesh Hydroxyapatite combined with demineralized bone matrix Titanium-alloy discs coated with a calcium phosphate and BMP-2 HA+BMP-2 Porous calcium phosphate ceramic Hydroxyapatite and beta-tricalcium phosphate Calcium phosphate ceramic Biphasic calcium phosphate (BCP) Porous Ti6Al4V material with an octacalcium phosphate (OCP) coating 3D printed bioceramic Calcium phosphate (Ca-P) coatings on porous titanium implants Octacalcium phosphate (OCP) coating on Ti6Al4V, HA, BCP, and PEGT-PBT copolymer HA/BCP Hydroxyapatite/ biphasic calcium phosphate Coralline porous ceramics Nano-sized CaP Porous hydroxyapatite Porous hydroxyapatite Porous hydroxyapatite Porous hydroxyapatite/poly-DL-lactide Porous calcium phosphate ceramics (HA/TCP) BSP/HA


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hydrogels for 3 wks (Appendix Table), and deproteinized bovine bone coated with a layer of calcium phosphate including BMP2 for 5 wks (Appendix Table)—all have demonstrated new ectopic bone formation. Although the effects of PDGF on MSC recruitment to sites of injury have been well-documented, the ability of PDGF to stimulate ectopic bone formation in intramuscular defects has not been fully demonstrated. Studies in immunocompromised mice have revealed that PDGF (Ranly et al., 2005) and platelet-rich plasma (Ranly et al., 2007) decrease the osteoinductivity of DBM. Furthermore, a recent study comparing the osteoinductive effects of a hydroxyapatite/TCP scaffold in combination with enamel matrix derivative or PDGF displayed no signs of osteoinduction in a murine thigh muscle model in mice (Chan et al., 2011).

Experimental design Of osteoinductive materials To maximize the potential of osteoinductive biomaterials, a bone graft should be capable of (1) recruiting mesenchymaltype osteoprogenitor cells, (2) transforming an undifferentiated mesenchymal cell into a mature, bone-forming osteoblast, and (3) inducing new bone formation when implanted into intramuscular locations. With the advances in modern biology, it is possible to characterize each of these 3 principles individually. The ability to test migration patterns, growth factor and cytokine release, as well as live behavioral cell migration microscopy has influenced our ability to describe recruitment of mesenchymal progenitor cells accurately. Many chemokine assays—such as transwell assays, chemotaxis assays, Boyden chambers, invasion assays, and quantitation assays with live video-microscopy— need to be fully utilized to investigate the migration patterns of osteoblast precursors accurately. Furthermore, the release of potent cytokines such as BMPs should be investigated with realtime PCR and ELISA to determine how biomaterials affect protein release of cells attached to their surfaces. If a material is truly osteoinductive, it should be capable of inducing differentiation of MSCs without the use of in vitro osteogenic differentiation media. The early expression of the transcription factors Runx2 and OSX as well as osteoblast differentiation markers including collagen 1 and OC should be increased via detection by real-time PCR. Furthermore, ALP assays and mineralization staining such as von Kossa and alizarin red staining should be used. Last, the implantation of bone grafts into extraskeletal locations such as muscle should be capable of bone formation in vivo. The successful combination of these 3 elements will guide the future direction of emerging biomaterials in bone regeneration.

Summary of experimental protocol (1) In vitro MSC migration assay. (2) In vitro MSC differentiation to mature osteoblasts. Realtime PCR for Runx2, ALP, Col1, and OCN. ALP assay and calcium assay (either alizarin red or von Kossa staining). (3) In vivo implantation of material injected into muscle. Bone formation should be observed in rats after 3 wks.

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Concluding remarks and perspectives The perspectives discussed herein demonstrate the importance of a full understanding of biological phenomena for the application of osteoinductive biomaterials. Over the past century, major advances in biomaterials have been made, and the future field of bone grafts poses many challenges to meet the increasing demands on healthcare. Our understanding of mesenchymal cell behavior (Shimono et al., 2011) will provide clues to the development of new material designs with osteoinductive ability. This will greatly widen the design parameters for the next generation of biomaterials and answer many important questions that have been raised. It is currently unknown what factors guide MSCs to sites of damage through transmigration across the endothelium. The homing of MSCs has gained tremendous awareness in stem cell therapy. MSC homing is a rapid process defined by the ability of MSCs to migrate through the blood, across the endothelial vasculature, to different organs and to their bone marrow niches (Quesenberry and Becker, 1998; Lapidot et al., 2005). It has been proposed that injured tissues expressing specific ligands facilitate trafficking and infiltration of MSCs to sites of injury, much like recruitment of leukocytes to sites of inflammation (Sordi, 2009). Leading candidates for MSC migration include chemokine SDF-1 and its interaction with CXCR4 on MSC surfaces, which allow them to relocate and regulate trafficking of MSCs to damaged tissues following a SDF1 gradient (Carbajal et al., 2010). These studies, together with studies on hematopoietic cell migration, may provide the platform needed to facilitate the identification of key factors involved in MSC migration (Tang et al., 2009; O’Sullivan et al., 2011). Furthermore, their interactions with cells from the immune system during inflammation could have an impact on the way MSCs contribute to the repair process in recipients in vivo (Ohtaki et al., 2008; Constantin et al., 2009). The importance of containing chemotactic factors for MSC migration during regenerative processes is of paramount significance. Homing MSCs represent a powerful source of multipotential cells capable of repairing damaged tissues and organs, and their migration to sites of injury is critical for future osteoinductive biomaterials. The ability of osteoinductive materials themselves to recruit MSCs also raises some interesting questions. Some osteoinductive materials lack osteoinductive factors but are able to recruit and differentiate MSCs into osteoblasts in vivo. It is plausible that MSC recruitment in vivo occurs via an indirect effect by which a random MSC in contact with an osteoconductive surface releases osteoinductive factors, thereby indirectly recruiting osteoprogenitor cells. These conditions, although beneficial, need to be further investigated. The field of mesenchymal differentiation to chondrocytes and osteoblasts also draws interest. It is still unclear what governs endochondral vs. intramembranous ossification during implanted defects in vivo (Chan et al., 2009). Implantation with certain materials such as TCP undergoes endochondral ossification, whereas implantation with BMP2 appears to skip the chondrocyte phase and results in intramembranous ossification. A further understanding of the regulatory mechanisms of MSCs to


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both cell types will enhance our ability to generate more predictable results (Mendez-Ferrer et al., 2010; Oldershaw et al., 2010). The ability of compressive force to induce MSC differentiation needs further clarification as well. MSCs that are treated with mechanical compressive force or static stress can induce the differentiation of MSCs into osteoblasts and chondroblasts (Yanagisawa et al., 2007; Kim et al., 2010). The possibility of constant friction and loading on muscles injected with bone grafts may play a role in osteoblast differentiation. The future field of osteoinductive biomaterials faces many challenges to meet the coming demand for bone-grafting procedures worldwide. These guidelines will ultimately facilitate their future testing and provide better long-term design for leading osteoinductive biomaterials.

Acknowledgments The study was funded by the ITI Foundation for the Promotion of Implantology (586_2008), Basel, Switzerland. The authors declare no conflicts of interest with respect to the research, authorship, and/or publication of this article.

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