TISSUE ENGINEERING: Part B Volume 20, Number 5, 2014 ª Mary Ann Liebert, Inc. DOI: 10.1089/ten.teb.2013.0731

Coculture in Musculoskeletal Tissue Regeneration Gun-Il Im, MD

Most tissues in the body are made up of more than one cell type. For successful tissue regeneration, it is essential to simulate the natural conditions of the cellular environment as much as possible. In a coculture system, two or more cell types are brought together, interact, and communicate in the same culture environment. The coculture system provides a powerful in vitro tool in research on cell-to-cell communications, repair, and regeneration. This review provides an overview on recent studies on general platforms and applications of coculture systems to enhance musculoskeletal regeneration, with a particular focus on osteogenesis, chondrogensis, and angiogenesis.

Definition and Methods of Coculture

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issue is a three-dimensional (3D) structure that consists of cells and the extracellular matrix (ECM). Most tissues in the body are made up of more than one cell type. A living tissue constantly interacts with the surrounding cells and the ECM. This interaction influences the proliferation and differentiation of cells inside the tissue and organ.1,2 Coculture is one of the in vitro methods to reproduce these in vivo interactions. In a coculture system, two or more cell types are brought together, interact, and communicate in the same environment. The coculture system provides a powerful in vitro means for research on cell-to-cell communications, repair, and regeneration. The first reported coculture study investigated heterologous communication between rat ovarian granulosa cells and mouse myocardial cells in 1978. In the study, it was found that these heterologous cells communicate by means of gap junctions, a cell-to-cell contact-dependent mechanism.3 Cells can communicate with each other through two mechanisms: (i) direct interaction between adjacent cells by physical contact, and (ii) indirect interaction through soluble molecules secreted from other cells. A coculture system can be either two-dimensional (2D) or 3D. In a 2D system, the direct contact culture employs the cell-to-cell mixture. The cell-to-cell mixture is an inexpensive and technically simple method. However, reisolation of the cells after culture is difficult. In addition, direct contact with cells can transform the function and phenotype.4,5 In a noncontact culture, the culture medium is shared between different cells on a separated plate (transwell). The indirect noncontact coculture system is used to study paracrine interactions between distinct cell populations. The physically distinct locations of the cells make it possible to detect the individual phenotype changes and gene expression of each cell type.2,6–9 In this

system, a porous membrane can be used, which allows passage of medium, but not cells.10 Another approach uses preconditioned media obtained from one type of cell that may be used to culture other types of cells.11 Whereas metabolic effects on the other types of cells are easily identified through this method, spatial interactions are difficult to determine in this indirect noncontact system. Whereas the low cost and simplicity of 2D systems allow for high-throughput assay,4 the reproduction of complicated in vivo function of cells is difficult.12 Three-dimensional systems also include contact culture (spheroid, micromass, and scaffold) and noncontact culture (porous membrane). The spheroid method can be used to investigate the effect of interaction between the cells13 and to create complex structures such as blood vessels.4,14 As in 2D systems, cell-to-cell contact can alter cell behavior.4,15 The porous membrane method, which does not permit cell contact, is used to investigate the influence of molecular cross talk16,17 and cell differentiation under the influence of cytokines (Fig. 1). These various cocultures have been used to promote cell differentiation in the field of skeletal tissue engineering. The purpose of this review is to provide a general overview on recent studies on the use of coculture to regenerate bone and cartilage. Cocultures for Chondrogenesis

Cartilage is a unique tissue that consists of one cell type, that is, chondrocytes. Cell-to-cell interactions between chondrocytes and other cells chiefly take place at the border of cartilage. In osteoarthritis (OA) research, coculture methods have been employed to investigate the development of OA by interactions between chondrocytes and synovial cells or between chondrocytes and osteogenic cells.8,18,19 Recent studies

Department of Orthopaedics, Dongguk University Ilsan Hospital, Goyang, Korea.

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FIG. 1. Concept of (A) two-dimensional (2D), (B) three-dimensional (3D), direct, and indirect contact culture. ECM, extra-cellular matrix. Color images available online at www.liebertpub.com/teb have been more dedicated to the induction of chondrogenesis from the mesenchymal stem cell (MSC)20,21 or from induced pluripotent stem cell (iPSC) or embryonic stem cell (ESC)22 by coculture (Table 1). Cocultures to induce chondrogenesis from MSC

Of late, cell-based therapy has become a mainstay strategy in cartilage repair. Chondrocytes expanded in in vitro culture are the most commonly used cell sources. However, as the proliferation potential of autologous chondrocytes decrease with increasing age of donors and passaging of cultured chondrocytes,23,24 human MSCs (hMSCs) have risen as alternative cell sources. Coculture with chondrocytes effectively induces chondrogenic differentiation of hMSCs.25 Paracrine signaling from soluble chondrogenic factors provided by chondrocytes can play an important role in inducing chondrogenesis of MSCs. When MSCs were seeded on the well and chondrocytes pellets were cultured in upper inserts, endogenous inductive factors from juvenile articular chondrocytes (ACs) drive MSCs into chondrogenic differentiation in the absence of direct physical contact and exogenous stimulators.26 The indirect culture with rib chondrocytes worked to induce chondrogenic differentiation of MSCs in both monolayer transwell culture and the 3D poly-lactic-co-glycolic acid (PLGA) scaffold.27 Cartilage explant secreted factors also induce chondrogenic differentiation of MSCs in a transwell system.28 In vivo chondrogenesis of bone marrow stromal cells (BMSCs) cocultured with chondrocytes on a biodegradable

scaffold was explored in a porcine study. Porcine BMSCs were mixed with ACs isolated from the porcine knee joint at a ratio of 1:1. The mixed cells were seeded onto the polyglycolic acid (PGA) scaffold and planted subcutaneously into nude mice. After 8 weeks of in vivo implantation, the constructs in both the coculture group and chondrocyte group formed cartilage-like tissue with a typical histological structure and ECM similar to those of the normal cartilage. The GAG content and compressive modulus of the coculture group reached over 80% of those of the chondrocyte group. On the other hand, the constructs of the BMSC group gradually shrunk after in vivo implantation without a typical cartilage-like tissue formation. This study shows that cocultured BMSC-chondrocyte-PGA constructs can form mature cartilage-like tissue in a subcutaneous nonchondrogenesis environment.29 When porcine ACs and BMSCs at the ratio of 3:7 were cotransplantated in the subcutaneous space, all the specimens in the cotransplantation group and chondrocyte group still formed homogeneous cartilage-like tissue after 8 weeks with a typical histological structure and specific matrix deposition similar to normal cartilage.30 Whereas chondrogenesis induced from MSCs with the use of growth factors can lead to hypertrophy and calcification, cocultures of ACs and MSCs have been proposed to avoid these problems. Paracrine factors released by ACs are believed to induce chondrogenesis, while inhibiting hypertrophy of MSCs in the coculture.31–34 When allogenic rat articular cartilage and rat MSCs were embedded in the alginate bead coculture system without addition of external growth factors, sustained expression of SOX9 from MSCs

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Table 1. Coculture for Chondrogenic Differentiation Cell type Synovial cells, chondrocytes Osteoblasts, chondrocytes hiPSCs or hESCs, chondrocytes hMSCs, chondrocytes Bovine bone marrow MSCs, bovine chondrocytes Goat bone marrow MSCs, goat chondrocytes Rat BMSCs, rat cartilage chip Human bone marrow MSCs, hACs Bovine bone marrow MSCs, bovine ACs hESCs, chondrocytes

Porcine bone marrow MSCs, chondrocytes TGF-b1-transduced hiPSCs, chondrocytes

Method Porcine chondrocytes in high-density pellet cultures with synovial fibroblast cell lines. It showed typical immature hyaline cartilage properties (expression of ECM components such as type II collagen). Sequential coculturing model that permits cell-to-cell contact and paracrine interaction between osteoblasts and chondrocytes in 3D culture. It was used to determine the effects of coculture on the phenotypic maintenance of osteoblasts and chondrocytes. Induce specific chondrogenic differentiation.

References 19 8

22

In the coculture of chondrocytes with hMSCs, expression of chondrogenic gene increased and chondrogenic matrix accumulated. Production of CDMD cells that best support neocartilage development. CDMD cells grown in a 3D hydrogel system developed into neocartilage.

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Transwell and PLGA scaffold coculture system promoted the chondrogenic differentiation of the goat MSCs. Coculture system of rat articular cartilage and rat BMSCs/alginate beads without addition of external growth factors. Coculture of hBMSCs and hACs improved chondrogenesis and suppressed the hypertrophy of MSCs.

27

Transwell coculture system of hESCs/chondrocytes secreted ECM containing glycosaminoglycan and expressed SOX9 and type II collagen. Implanted constructs with cocultured hESCs expressed type II collagen, type I collagen, total collagen, and GAG. Mixed coculture of porcine bone marrow MSCs and chondrocytes in PGA scaffold. After 8 weeks of subcutaneous implantation in mice, formation of tissue similar to normal cartilage Coculture in alginate matrix. Increased expression of cartilage-related genes. More new cartilage formed in the coculture with chondrocytes than those without chondrocytes from in vivo implantation.

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31 32–34

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CDMD, coculture-driven MSC differentiated; TGF, transforming growth factor; ECM, extracellular matrix; 3D, three-dimensional; hMSC, human mesenchymal stem cell; hiPSC, human-induced pluripotent stem cell; ESC, embryonic stem cell; PLGA, poly-lacticco-glycolic acid; BMSC, bone marrow stromal cell; PGA, polyglycolic acid; AC, articular chondrocyte.

was observed at an early stage, which indicated chondrogenic induction. Late stage repression of collagen X indicated prehypertrophic arrest of differentiation.31 In another study, hMSCs were differentiated in a chondrogenic medium conditioned by parallel culture with hAC pellets, or hMSCs were mixed in the same pellet with the hACs (1:1 or 1:2 ratio) and cultured for 6 weeks. The gene expression ratio of COL10A1 to COL2A1 and of Indian hedgehog (IHH) to COL2A1 was significantly reduced in hMSCs that underwent differentiation in the hAC-conditioned medium, and less type X collagen protein was deposited relative to type II collagen. The alkaline phosphatase (ALP) activity was significantly lower in the cells differentiated in a conditioned medium. In mixed hAC/hMSC pellets, suppression of ALP was dose dependent. Chondrocytes secreted the parathyroid hormone-related protein (PTHrP) throughout the culture period, whereas the PTHrP was downregulated and IHH upregulated in control MSCs after 2–3 weeks of chondrogenesis. The main inhibitory effects seen with the hAC-conditioned medium could be reproduced by PTHrP supplementation of an unconditioned medium.32 Bian et al. also tested whether the addition of chondrocytes to MSC cultures affects MSC hypertrophy when cultured in hya-

luronic acid hydrogels. The construct with mixed cell populations (hMSCs and human chondrocytes at a ratio of 4:1) exhibited a lower deposition of collagen X than in the constructs seeded with MSCs alone.34 Growth factors or mechanical stimuli can accelerate chondrogenic differentiation of MSCs in coculture. Coculture of synovial MSCs with transforming growth factor (TGF)-b3-transfected ACs enhances chondrogenic differentiation from MSCs.35 Mechanical stimuli potentially induces the differentiation of MSCs into a chondrocytic cell type without using biochemical agents. When MSCs and chondrocytes were encapsulated into alginate beads and cocultured, stimulation with higher magnitudes of intermittent hydrostatic pressure ( ‡ 0.10 MPa) affected the proliferation and differentiation of cocultured MSCs.36 Cocultures to induce chondrogenesis from ESC or iPSC

hESCs and hiPSCs have unlimited capacity for selfrenewal. These cells are capable of differentiating three germ layer lineages. The ESC has been investigated as an unlimited cell source for chondrocytes in articular cartilage

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repair. When hepatic cells (HEPA-1C1c7) were cocultured with ES-derived cells (ESDCs) in 3D bilayered hydrogels, the coculture significantly enhanced specific chondrogenic differentiation of ESDCs.37 Coculture of hiPSCs or hESCs with chondrocytes induces specific chondrogenic differentiation of hiPSCs or ESCs.22 Both the transwell coculture system and the implanted constructs of hESCs/chondrocytes induced chondrogenic differentiation, whereas a coculture with fibroblasts failed to induce chondrogenic differentiation.38 TGF-b1-transduced hiPSCs were cocultured with chondrocytes in the alginate matrix. Cartilage-related genes, such as collagen II, aggrecan, and cartilage oligomeric matrix protein (COMP), increased while vascular endothelial growth factor (VEGF), a degenerative cartilage marker, decreased. An in vivo implantation study revealed that the coculture of TGF-b1-transfected iPSCs with chondrocytes in the alginate matrix produced more new cartilage than those without the chondrocyte coculture.39 Cocultures to induce chondrogenesis from passaged chondrocytes

When chondrocytes are passaged for proliferation, they gradually lose the biochemical properties of chondrocytes. A coculture with fresh, unpassaged chondrocytes can be used to induce redifferentiation of passaged cells. Coculture of primary and passaged bovine chondrocytes grown on filter inserts upregulated type II collagen and SOX9, while decreasing type I collagen gene expression in the passaged chondrocytes. This coculture model clearly showed that the passaged chondrocytes had the potential to redifferentiate when cocultured with the primary chondrocytes.40

Coculture for Osteogenic Differentiation

Whereas osteogenic differentiation of MSCs is induced by ascorbic acid, dexamethasone, and b-glycerol phosphate,41 coculture with osteoblasts/osteocytes or endothelial cells (ECs) enhances osteogenic differentiation from MSCs. The osteogenic differentiation of MSCs comprises three distinct steps: early cell differentiation at the peak in a number of cells, the expression of a type I collagen, and the final step is expression of osteocalcin and osteopontin.42 The recent studies on coculture for osteogenesis are summarized in Table 2. Interaction between MSC and osteoblast/osteocyte

When the synergistic relationship between osteoblasts/osteocytes and MSCs was examined by transwell coculture, intracellular ALP peaked earlier and a greater amount of calcium deposited when MSCs were cocultured with osteocytes rather than osteoblasts, suggesting that an osteocyte is more influential than an osteoblast in stimulating osteogenesis in MSCs. Osteoblasts initially induced an increase in the number of MSCs, but ultimately regulated MSC differentiation down the same pathway. When MSCs were simultaneously exposed to factors from both osteoblasts and osteocytes, the osteogenic effect was higher than exposure to either cell type alone, suggesting a functional relationship in the osteocyte–osteoblast network.43 Osteoinductive factors are necessary for the osteogenic effect of osteoblasts on MSCs. In a mixed coculture of murine MSC cell line C3H10T1/2 and murine osteoblast cell line MC3T3-E1 (clone 14) either with or without osteogenic supplements in a culture medium, osteoblasts provided little to osteogenic differentiation of MSCs without osteogenic supplementation.44

Table 2. Coculture for Osteogenic Differentiation Cell type

Method

Mouse MSCs, osteocytes, Osteogenic differentiation of MSCs, as characterized by alkaline or osteoblasts phosphatase activity and mineralization, was greater when MSCs were cocultured with osteocytes rather than osteoblasts. hMSCs, osteoclast-like Adipogenic differentiation of hMSCs was suppressed by the presence cells, or osteoblasts of osteoclasts. Murine MSCs, Cocultures required at least one osteoinductive factor to induce or enhance osteoblasts the osteogenic differentiation in MSCs. Human bone marrow Human bone marrow MSCs were cocultured with HUVECs using a 3D spheroid MSCs, HUVECs coculture system. HUVECs inhibited adipogenic differentiation and the proliferation of MSCs in 3D cocultures, indicating that HUVECs suppressed MSC cycling and selectively promoted osteogenic differentiation in 3D. Human bone marrow Coculture of hMSCs with HUVECs induced osteogenic and angiogenic MSCs, HUVECs differentiation. MSCs, MSCb-TCP scaffold of coculturing MSCs and ECs promoted repair bone defects. derived ECs hMSCs, HUVECs b-TCP scaffold of coculturing MSCs and ECs enhanced osteogenic differentiation. Mouse bone marrow Low dose of HSPCs in coculture with MSCs enhanced osteogenesis. MSCs, HSPCs hASCs, VECs Enhanced osteogenic differentiation of hASCs. hASCs, DM cells DM-derived BMP-2 paracrine stimulation appears to play a key role for hASC-mediated repair.

References 43 45 44 51

52 50 57 60 63–65 67

VEC, vascular endothelial cell; HUVEC, human umbilical vein endothelial cell; b-TCP, beta-tricalcium phosphate; HSPC, hematopoietic progenitor cell; hASC, human adipose stem cell; DM, dura mater; BMP, bone morphogenetic protein.

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Osteoclasts may have a role in promoting osteogenic differentiation of MSCs. Human monocytes and hMSCs, which were differentiated into osteoclast-like cells and osteoblasts, respectively, were cocultivated in a culture system made of mineralized collagen I. Transwell inserts spatially separated the cell types, but allowed exchange of soluble factors. The osteo-induced hMSC showed an increased gene expression and activity of ALP when cocultivated with differentiating osteoclasts. Adipogenic differentiation of hMSCs was suppressed by the presence of osteoclasts. There was a major decrease in the adipocyte cell numbers and a decrease in gene expression of adipogenic markers. The formation of multinucleated osteoclasts seems to be decreased in the presence of osteo-induced hMSCs.45 In another study, when nonadherent rat bone marrow cells (hematopoietic cells) or their conditioned media were added to rat BMSCs, the number of osteoprogenitors increased.46 Interaction between MSC and EC

MSCs reside in a perivascular niche, closely related to pericytes.47–50 When human umbilical vein endothelial cells (HUVECs) were cocultured with hMSCs in a spheroid coculture system, HUVECs selectively promoted osteogenic differentiation of MSCs, while inhibiting adipogenic differentiation.51,52 Proliferation of MSCs was also enhanced by coculture with ECs.53 Beta-tricalcium phosphate (b-TCP) is an osteoconductive bone substitute with good biodegradability and biocompatibility.54,55 A b-TCP scaffold can provide a vehicle for coculturing MSCs and ECs to repair the bone defects.56 HUVECs enhanced the early osteogenic differentiation of MSCs in cocultures inside the b-TCP scaffold.57 Interaction between MSC and hematopoietic stem cell

Hematopoietic progenitor cells (HSPCs) are primarily located at the endosteal surface of trabecular bone.58,59 The HPSC and bone marrow MSC have close interaction and mutually influence the development of each other. Coculture of HSPCs and MSCs enhances the osteogenic differentiation of MSCs through both cell-to-cell interactions and paracrine signaling.60 Interaction between adipose stem cells and other cells

Adipose stem cells (ASCs) also have a strong multidifferentiation ability, making them an idea cell source in orthopedic tissue regeneration.61 ASCs secrete VEGF, which has vital importance for osteogenesis.62 When hASCs and HUVECs were cocultured, osteogenesis of hASCs was enhanced.63–65 Human osteoblasts exposed to tumor necrosis factor-alpha (TNF-a) induced a significantly greater osteogenic differentiation of ASCs than did the osteoblasts without TNF-a treatment.66 Coculture of hASCs and dura mater (DM) cells also promoted osteogenesis from hASCs. Cell-to-cell interactions between ASCs and host DM cells were critical for the healing of calvarial defects. DM-derived bone morphogenetic protein signaling was partly necessary in hASC-mediated calvarial healings.67 Cocultures to Enhance Angiogenesis

One of the main priorities in tissue engineering is to prevent the death of implanted cells. Rapid vascularization

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of a construct is necessary to ensure the survival of grafted cells.68,69 Angiogenesis includes four distinct steps: degradation of basement membrane, proliferation and invasion into the subjacent ECM, maturation of nascent vessels, and pruning of regression vasculature.70 Angiogenesis can be enhanced by a close interaction between ECs and osteoblasts.10,71,72 The cross talk between two types of cells also includes diffusible growth factors secreted by osteoblasts.72,73 The human osteoblast supports cell proliferation and spontaneous formation of multiple tube-like structures by HUVECs.74 The results of recent studies are summarized in Table 3. Molecules related to angiogenesis in coculture

VEGF is a key factor in physiological and pathological angiogenesis. VEGF plays an important role in the intercommunication between osteoblasts and ECs.75 In a direct contact coculture model between human osteoprogenitors (HOPs) and HUVECs, specific tubular structures were formed. VEGF gene expression was upregulated in the cocultured HOPs, and both Flt-1 and VEGF-receptor 2 (KDR) gene expressions increased in cocultured HUVECs. However, the cell rearrangement observed in the coculture was promoted by a combination of soluble chemoattractive factors and not by VEGF alone.76 In a 2D coculture of human bone marrow MSCs and HUVECs, expression of VEGF was upregulated in cocultured MSCs. Meanwhile, KDR and urokinasetype plasminogen activator (uPA) were upregulated in cocultured HUVECs. Neutralization of VEGF suppressed the migration and the rearrangement of the cells and downregulated the expression of uPA and its receptor. Blocking of vascular endothelial cadherin did not influence the migration of cocultured HUVECs, but it did suppress the self-assembled network formation.76,77 Sonic hedgehog (Shh) also promotes angiogenesis in the coculture of ECs and primary osteoblasts. Shh leads to a massive increase in microvessel-like structures in the cocultures compared with untreated ones. Increased formation of angiogenic structures seems to correlate with the upregulation of VEGF or angiopoietins (Ang-1 and Ang-2) at both the mRNA and protein levels. Treatment with cyclopamine, an inhibitor of hedgehog signaling, prevented the formation of microvessel-like structures in the cocultures.78 When cells were treated either with VEGF or Shh for 1 week, both factors led to an increase in the formation of microvessel-like structures. Compared with VEGF, Shh also stimulated the expression and secretion of Angs, which were detected after 24 h of treatment. Moreover, smooth muscle cell-related markers, including a-smooth muscle actin, desmin, and myocardin as well as basement membrane components were clearly upregulated in response to Shh treatment compared with VEGF.79 Other factors affecting angiogenesis in a coculture

Endothelial progenitor cells (EPCs) from umbilical cord blood form more stable blood vessels than EPCs from adult blood. Whereas adult peripheral blood EPCs form blood vessels that are unstable and regress within 3 weeks, umbilical cord blood EPCs form normal-functioning blood vessels that last for more than 4 months.80 In cocultures of MSCs and ECs, differentiation is affected by different culture media. The osteogenic differentiation

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Table 3. Coculture for Angiogenesis Cell type Fibroblasts, ECs BMSC-derived osteoblasts, ECs hASCs, ECs Human bone marrow MSCs, OECs HOPs, HUVECs Human bone marrow MSCs, HUVECs

Method Enhanced the angiogenesis of ECs BMSC-derived osteoblasts and ECs compound with CS/HA porous scaffold promoted bone formation and vascularization in bone defect and accelerated the healing of bone defect. In coculture conditions with ASCs and ECs under treatment with growth factors, ASC significantly increased EC viability, migration, and tube formation. Enhanced the differentiation of MSCs to osteoblastic lineages, involved in vessel stabilization.

Enhanced both primary angiogenesis events and osteoblastic differentiation. Cocultures upregulated the expression of VEGF in cocultured hMSCs; VEGF then activated uPA in cocultured HUVECs, which might be responsible for initiating the migration and the self-assembled network formation with the participation of VE-cad. CE-EPCs, HUVECs, Local VEGF and/or PlGF expression promoted vasculogenesis; VEGF plays or hMECs a role in EPC recruitment and subsequent formation of functional vessels. MSCs, human Normoxic coculture model containing cells from clinically relevant sources sustained ECFCs simultaneous endothelial, smooth muscle, and osteogenic differentiation. HUVECs, human SCPP promoted angiogenesis and bone regeneration in in vitro and in vivo coculture osteoblasts model of HUVECs and osteoblasts. Human OECs, OECs formed vascular structures at a similar density as those arising from the host. osteoblasts In addition to the known paracrine activity of osteoblasts to cocultured OECs, osteoblasts provide additional structural support for OEC-derived vessels.

References 88,89 84 92 81 76 77

82 83 85 86

CS/HA, chitosan/hydroxyapatite; OEC, outgrowth endothelial cell; HOP, human osteoprogenitor; CE-EPCs, culture expanded endothelial progenitor cells; hMEC, human microvascular endothelial cell; ECFC, endothelial colony forming cell; SCPP, strontium-doped calcium polyphosphate; VEGF, vascular endothelial growth factor; PlGF, placental growth factor; uPA, urokinase-type plasminogen activator; VE-cad, vascular endothelial-cadherin.

medium (ODM) was more suitable for the differentiation of MSCs to osteoblastic lineages in the cocultures, whereas the endothelial cell growth medium-2 (EGM2) formed more microvessel-like structures compared with cocultures in the ODM, as shown by immunofluorescence staining for CD31, an endothelial marker.81 Whereas hypoxia is considered a strong inducer for the creation of new vasculature,82 hypoxia impedes vasculogenesis in the coculture of human endothelial colony forming cells (ECFCs) and MSCs. Whereas the normoxic condition in cocultures of MSCs and ECFCs supported the formation and maintenance of prevascular structures, including organized CD31-positive cells embraced by differentiated mural cells, these structures failed to form in hypoxic conditions (5% O2).83 The appropriate scaffold promotes angiogenesis in a coculture. Coculture of BMSC-derived osteoblasts and ECs with the chitosan/hydroxyapatite (CS/HA) scaffold promotes bone formation and vascularization in bone defect. BMSC-derived osteoblasts and ECs (1:1) seeded in the CS/ HA porous scaffold promoted healing in radial defects of rats.84 Strontium-doped calcium polyphosphate (SCPP) also promotes angiogenesis and bone regeneration in an in vitro and in vivo coculture model of the HUVEC and osteoblast. The optimal ratio of the HUVEC and osteoblast cocultured model for in vitro angiogenesis was 5:1. Compared with those in calcium polyphosphate or hydroxyapatite scaffold, the in vivo formation of tube-like structures and the expression of platelet EC adhesion molecules in the cocultured model were better in the SCPP scaffold.85 When human outgrowth endothelial cells (OECs) isolated from human peripheral blood were cocultured with primary human os-

teoblasts, they formed perfused vascular structures within a starch–poly(caprolactone) biomaterial after 48 h following in vivo subcutaneous implantation in mice. OECs formed vascular structures at a similar density as those arising from the host. In addition to the known paracrine activity of osteoblasts to cocultured OECs, osteoblasts offer an additional structural support for OEC-derived vessels, perhaps playing a pericyte-like role.86 Coculture with other cells and ECs to promote angiogenesis

An in vitro cell coculture of fibroblasts and ECs significantly enhances the angiogenesis of ECs.12,87–92 Paracrine effects from fibroblasts, including the secretion of basic fibroblast growth factor and VEGF, play an important role in cellular communication.90,91 In a coculture system of ASCs with ECs, ASCs release angiogenic growth factors, including VEGF, capable of inducing EC differentiation.92 Conclusion and Perspective

Coculture provides a powerful tool for understanding cellular interactions with other cell types as well as enhancing differentiation into certain cell lineages for tissue repair. Both cell-to-cell contact and paracrine mechanism work in the coculture system. Whereas the use of growth factors and small molecules has been used to promote osteogenesis, chondrogenesis, and angiogenesis, the use of coculture may provide a more natural and effective way to serve the purpose. On the other hand, molecular and humoral mechanisms underlying enhanced differentiation have not been studied in detail so far. Investigation of secreted

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factors elevated during the coculture and tracking related to the signal transduction pathways in the cell can provide a cue on the mechanism. Whereas coculture can offer a more integral signal for differentiation to a certain lineage, the very characteristics of coculture prevent the standardization of the technique because cells vary from one donor to another. In addition, using cells to induce differentiation can be economically challenging and unpractical in a large-scale culture. The understanding on molecular process occurring in a coculture process can possibly provide a means to mimic the coculture condition in a more controllable and standardized way. Acknowledgments

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12. 13. 14.

This work was supported by the Bio and Medical Technology Development Program of the National Research Foundation (NRF) funded by the Korean government (MEST) (2012M3A9B4028566).

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Disclosure Statement

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No competing financial interests exist. References

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Address correspondence to: Gun-Il Im, MD Department of Orthopaedics Dongguk University Ilsan Hospital Goyang 410-773 Korea E-mail: [email protected] Received: December 2, 2013 Accepted: February 18, 2014 Online Publication Date: March 21, 2014