Mesenchymal Stem Cells in Bone Tissue Regeneration and Application to Bone Healing

This synoptic study gives a concise overview of current knowledge of bone healing, the role of mesenchymal stem cells in bone tissue regeneration and contemporary possibilities of supporting regeneration of damaged bone. Attention of research concerning the healing of fractures with extensive loss of bone tissue following trauma, the treatment of belatedly healing or non-healing fractures or the healing of segmental bone defects following tumour resection, is focused on development of three-dimensional scaffolds planted with mesenchymal stem cells that might be used for reconstruction of such large bone lesions. Presented are possibilities of transplantation of mesenchymal stem cells combined with biomaterials into bone defects, including the results of our own experimental studies dealing with the use of stem cells in the treatment of damaged tissues of the musculoskeletal system in animal models. Tissue engineering, biomaterials, scaffold, segmental bone lesion, fracture healing, growth factors, review Contemporary research currently focuses on the use of mesenchymal stem cells (MSCs) for the purpose of regeneration of damaged tissues of the musculoskeletal system, such as the cartilage, bone, ligaments, muscles and tendons (Ahn et al. 2004; Arinzeh 2005; Arthur et al. 2009; Award et al. 2003; Chen et al. 2003; Dressler et al. 2005; Gál et al. 2007; Hoemann et al. 2005; Lee and Hui 2006; Plánka et al. 2007, Shirly et al. 2005; Kraus and Kirker-Head 2006; Nečas et al. 2008; Noel et al. 2002; Waese et al. 2008; Zaidi and Nixon 2007). Bone tissue is capable of regeneration, yet the natural bone healing process is in some cases insufficient. For example, excessive loss of bone due to trauma or tumour resection, non-healing fractures, metabolic diseases, arthrodesis, vertebral fusion, insufficient healing capacity due to systemic, local disease or age etc., present cases where bone regeneration using transplantation of MSCs alone or combined with biomaterials may bring the required result of successful healing of the particular bone defect (Cancedda et al. 2003, Caplan 2005; Drosse et al. 2008; Jančář et al. 2007; Kraus and Kirker-Head 2006; Nečas et al. 2008; Salgado et al. 2006; Slater et al. 2008; Viateau et al. 2007). The aim of this work was to give a concise overview of existing knowledge from experimental studies on regeneration of bone tissue using methods of tissue engineering with the application of MSCs transplantation; and concurrently, to inform on the results of our own research studies in relation to MSCs transplantation into the tissues of the musculoskeletal system in animal models (miniature pig, New Zealand white rabbit). ACTA VET. BRNO 2009, 78: 635-642; doi:10.2754/avb200978040635 Address for correspondence: MVDr. Michal Crha, Ph.D. Department of Surgery and Orthopaedics Small Animal Clinic Faculty of Veterinary Medicine University of Veterinary and Pharmaceutical Sciences Brno Palackeho 1-3, 612 42 Brno, Czech Republic Phone: +420 541 562 362 E-mail: crham@vfu.cz http://www.vfu.cz/acta-vet/actavet.htm Role of MSCs in bone tissue regeneration When bone integrity is damaged (e.g. after fracture), under normal circumstances MSCs play an important role in its healing. MSCs are multipotent cells of mesodermal origin capable of differentiating into osteoblasts, chondrocytes, adipocytes, tenocytes and myoblasts (Heino and Hentunen 2008; Jaswal et al. 1997; Krampera et al. 2006; Marie and Fromigue 2006; Pittenger et al. 1999; Smith 2006). These cells are identified as marrow stromal cells (supporting the cells of bone marrow), because they are relatively abundant in the bone marrow which is their suitable source. Apart from bone marrow, they are found in the endosteum of the trabecular bone, and the periosteum (Stocum 2001). A limited source of MSCs is the fat tissue, funicle blood, muscle and synovial membrane (Yoo and Johnstone 1998; Bruder et al. 1997). MSCs are found in small quantities in peripheral blood and other tissues (Kuznetsov et al. 2001). They have been isolated e.g. from the liver, brain and pancreas (Porada et al. 2006). MSCs of the bone marrow and endosteum originate from the periosteum. During foetal development the calcified cartilage of endochondral bone is gradually reconstructed and vascularised, MSCs are transferred from the periosteum to the marrow cavity where MSCs are further differentiated into osteoblasts substituting bone cartilage, fibroblasts and adipocytes that form the supporting tissue of the bone marrow in formation. Concurrently, part of the MSC population in bone marrow remains unchanged and forms the source of undifferentiated stem cells (Stocum 2001). Bone regeneration is analogous to embryonic development of the skeleton. It is provided by a sum of cellular, humoral and mechanical factors involved in the new formation of bone in which MSCs play an important role. At the site of the fracture line the bone is damaged, which is accompanied by bleeding. Cytokines released from the damaged matrix of the bone and from degranulated thrombocytes form a mix of biologically active proteins, some of which affect MSCs chemotactically. MSCs from the periosteum and bone marrow are transferred to the location of bone damage where they continue to multiply and differentiate into osteoblastic, chondroblastic and fibroblastic lines of cells (Oe et al. 2007) responsible for the production of bone matter and cartilage that form a callus at the fracture site (Einhorn 1998; Carter et al. 1998). Recently published studies show that during bone injury, MSCs are flowed from bone marrow to peripheral blood. Through peripheral blood the originally distant MSCs are transferred to the site of bone injury where they reinforce the healing potential of local MSCs (Devine at al. 2002; Shirley et al. 2005). Bone morphogenic proteins (BMPs) play an important role during prenatal development and bone regeneration (Reddi 2000). They carry out the task of cytokines that fundamentally influence MSCs, as they can modify their differentiation (Edgar et al. 2007). Osteogenesis is the result of mutual interaction of individual types of BMPs, when e.g. BMP-2, -4, and -7 are responsible mainly for the induction of osteogenesis, whereas BMP-12, -13, and -14 are connected with cartilage formation (Reddi 2001; Li and Wozney 2001; Carter 2003). Methods of MSCs isolation are based on their ability to divide and adhere to the substrate or surface of the cultivation container (Caplan 1991; Stocum 2001). During cultivation and the passaging of cells obtained by bone marrow aspiration, MSCs may be separated by a change of the cultivation medium from the cells that do not possess the ability of adhesion and move freely in the cultivation solution (e.g. the line of haematopoietic stem cells, HSCs) (Stocum 2001). The results of experimental studies point out that during cultivation in vitro, MSCs may be directed toward transformation into lines of cells that are capable of producing bone matter by being exposed to the effects of a number of substances, such as the transforming growth factor (TGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), dexamethasone or glycerol phosphate, vitamin D and bone morphogenic protein 2 (BMP-2) (Pluhar 2004; Anitua et al. 2004a; 636

Contemporary research currently focuses on the use of mesenchymal stem cells (MSCs) for the purpose of regeneration of damaged tissues of the musculoskeletal system, such as the cartilage, bone, ligaments, muscles and tendons (Ahn et al. 2004;Arinzeh 2005;Arthur et al. 2009;Award et al. 2003;Chen et al. 2003;Dressler et al. 2005;Gál et al. 2007;Hoemann et al. 2005;Lee and Hui 2006;Plánka et al. 2007, Shirly et al. 2005;Kraus and Kirker-Head 2006;Nečas et al. 2008;Noel et al. 2002;Waese et al. 2008;Zaidi and Nixon 2007).Bone tissue is capable of regeneration, yet the natural bone healing process is in some cases insufficient.For example, excessive loss of bone due to trauma or tumour resection, non-healing fractures, metabolic diseases, arthrodesis, vertebral fusion, insufficient healing capacity due to systemic, local disease or age etc., present cases where bone regeneration using transplantation of MSCs alone or combined with biomaterials may bring the required result of successful healing of the particular bone defect (Cancedda et al. 2003, Caplan 2005;Drosse et al. 2008;Jančář et al. 2007;Kraus and Kirker-Head 2006;Nečas et al. 2008;Salgado et al. 2006;Slater et al. 2008;Viateau et al. 2007).The aim of this work was to give a concise overview of existing knowledge from experimental studies on regeneration of bone tissue using methods of tissue engineering with the application of MSCs transplantation; and concurrently, to inform on the results of our own research studies in relation to MSCs transplantation into the tissues of the musculoskeletal system in animal models (miniature pig, New Zealand white rabbit).

Role of MSCs in bone tissue regeneration
When bone integrity is damaged (e.g. after fracture), under normal circumstances MSCs play an important role in its healing.MSCs are multipotent cells of mesodermal origin capable of differentiating into osteoblasts, chondrocytes, adipocytes, tenocytes and myoblasts (Heino and Hentunen 2008;Jaswal et al. 1997;Krampera et al. 2006;Marie and Fromigue 2006;Pittenger et al. 1999;Smith 2006).These cells are identified as marrow stromal cells (supporting the cells of bone marrow), because they are relatively abundant in the bone marrow which is their suitable source.Apart from bone marrow, they are found in the endosteum of the trabecular bone, and the periosteum (Stocum 2001).A limited source of MSCs is the fat tissue, funicle blood, muscle and synovial membrane (Yoo and Johnstone 1998;Bruder et al. 1997).MSCs are found in small quantities in peripheral blood and other tissues (Kuznetsov et al. 2001).They have been isolated e.g. from the liver, brain and pancreas (Porada et al. 2006).
MSCs of the bone marrow and endosteum originate from the periosteum.During foetal development the calcified cartilage of endochondral bone is gradually reconstructed and vascularised, MSCs are transferred from the periosteum to the marrow cavity where MSCs are further differentiated into osteoblasts substituting bone cartilage, fibroblasts and adipocytes that form the supporting tissue of the bone marrow in formation.Concurrently, part of the MSC population in bone marrow remains unchanged and forms the source of undifferentiated stem cells (Stocum 2001).Bone regeneration is analogous to embryonic development of the skeleton.It is provided by a sum of cellular, humoral and mechanical factors involved in the new formation of bone in which MSCs play an important role.At the site of the fracture line the bone is damaged, which is accompanied by bleeding.Cytokines released from the damaged matrix of the bone and from degranulated thrombocytes form a mix of biologically active proteins, some of which affect MSCs chemotactically.MSCs from the periosteum and bone marrow are transferred to the location of bone damage where they continue to multiply and differentiate into osteoblastic, chondroblastic and fibroblastic lines of cells (Oe et al. 2007) responsible for the production of bone matter and cartilage that form a callus at the fracture site (Einhorn 1998;Carter et al. 1998).
Recently published studies show that during bone injury, MSCs are flowed from bone marrow to peripheral blood.Through peripheral blood the originally distant MSCs are transferred to the site of bone injury where they reinforce the healing potential of local MSCs (Devine at al. 2002;Shirley et al. 2005).Bone morphogenic proteins (BMPs) play an important role during prenatal development and bone regeneration (Reddi 2000).They carry out the task of cytokines that fundamentally influence MSCs, as they can modify their differentiation (Edgar et al. 2007).Osteogenesis is the result of mutual interaction of individual types of BMPs, when e.g.BMP-2, -4, and -7 are responsible mainly for the induction of osteogenesis, whereas BMP-12, -13, and -14 are connected with cartilage formation (Reddi 2001;Li and Wozney 2001;Carter 2003).
Methods of MSCs isolation are based on their ability to divide and adhere to the substrate or surface of the cultivation container (Caplan 1991;Stocum 2001).During cultivation and the passaging of cells obtained by bone marrow aspiration, MSCs may be separated by a change of the cultivation medium from the cells that do not possess the ability of adhesion and move freely in the cultivation solution (e.g. the line of haematopoietic stem cells, HSCs) (Stocum 2001).The results of experimental studies point out that during cultivation in vitro, MSCs may be directed toward transformation into lines of cells that are capable of producing bone matter by being exposed to the effects of a number of substances, such as the transforming growth factor (TGF), vascular endothelial growth factor (VEGF), insulin-like growth factor (IGF), dexamethasone or glycerol phosphate, vitamin D and bone morphogenic protein 2 (BMP-2) (Pluhar 2004;Anitua et al. 2004a;Carter 2003;Anitua et al. 2004b;Marx 2004, Pittenger et al. 1999, Prockop 1997).Experimental studies report other properties of MSCs, such as the ability to maintain the possibility of division after cryopreservation (Bruder 1997) or their immunotolerance.Some studies suggest that MSCs lack certain receptors on their surface which allows them to escape the T-cell component of immunity (Pittenger et al. 1999;Devine 2002).Other studies even point out immunosuppressive properties of MSCs after their transplantation (Devine et al. 2001;Bartholomew et al. 2002;Porada et al. 2006).It is assumed that due to these immunological properties, allogeneic MSCs might be used in transplantations as effectively as autogenous MSCs (Arinzeh et al. 2003;Kraus a Kirker-Head 2006).A possibility has even been described of xenogeneic transplantation of MSCs for the purpose of bone tissue regeneration in rabbits (Bruder et al. 1998b).

Contemporary strategies of supporting bone defect healing
Regeneration of damaged bone is related to several fundamental processes: osteogenesis, osteoinduction, osteoconduction and osteopromotion.Efforts are made to achieve the most effective way of regeneration possible, using the optimum combination of these four processes with the application of MSC implantation (Bruder and Fox 1999;Nečas et al. 2008, Viateau et al. 2007;Kirker-Head et al. 2007).The best example of the use of the osteogenic potential of transplanted cells is the autogenous spongeous bone graft (Kraus and Kirker-Head 2006).The collection location in dogs and cats is crista iliaca or tumerculum majus humeri, in horses it is sternum and alla ossis ilii (Johnson 2007).The disadvantage of graft collection is the necessity of preparing another operation field, which increases the material cost and lengthens the surgery time.A relative disadvantage is also the insufficient yield of a graft in small or old individuals.In humans, graft collection is moreover associated with subsequent pain at the graft collection location and higher morbidity (Beirne et al. 1996;Silber et al. 2003;Joshi and Kostakis 2004).Autogenous spongeous bone graft is considered the "golden standard" among tissue transplants supporting bone regeneration, but not always all aspects of the application of this graft are ideal for clinical practice.Attention of contemporary research is therefore directed to the finding of an optimum substitute for the standard bone grafts used.Promising results have been yielded by the application of BMPs combined with materials showing osetoconductive properties, such as deproteinised bone, some forms of demineralised bone matrix (DBM), synthetic collagene, hydroxyapatite, tricalcium phosphate, hydrogel based on hyaluronic acid and some synthetic polymers based on polyglycolic acid or polylactate (Cook et al. 1994;Gao et al. 1996;Boyan et al. 1999;Yamamoto et al. 1998;Horisaka et al. 1991;Hotz and Herr 1994).Commercial application of DNA recombinant technology allowed the synthesis of recombinant human bone morphogenic proteins (rh BMPs) using bacteria (e.g.Escherichia coli) (Vallejo et al. 2002).Production of the BMP has thus been substantially speeded up and facilitated, which contributed to its wider application in clinical practice.At present, rh BMP-2 (Genetics Institute, Boston, Massachusetts, USA) and rh BMP-7 Creative BioMolecules, Hopkinton, Massachusetts, USA) are commercially available on the U.S. market.Clinical application of rh BMPs has been described in humans in orthopaedic interventions (vertebral fusion, long bone defect healing, nonhealing fractures), in craniomaxillofacial surgery, and dentistry (Johnson and Urist 1998;Boyne 2001, Friedlaender et al. 2001;Burkus et al. 2002).The application of BMPs has yielded positive results in supporting bone regeneration, yet their exclusively osteoinductive property presents a certain strategic limitation.New bone formation is dependent on MSCs present at the location of bone damage that represent the source of osteogenic lines of cells capable of forming bone matter.In this sense, the strategy of using the osteogenic potential of MSCs transplanted into the bone defect appears promising.The subject of intensive research in the field of tissue engineering is the application of MSCs in combination with suitable scaffolds in order to achieve bone tissue regeneration.This would be a contribution for clinical practice in patients with extensive bone defects (tumour resection, traumatic injuries with bone loss, complicated fractures) or in cases of decreased healing ability of bone tissue (older age, osteoporosis) or genetic diseases of the skeleton (osteogenesis imperfecta) (Barry et al. 2001).

Strategies of transplantation of MSCs in combination with biomaterials
The primary aim of tissue engineering is the finding of suitable material biocompatible with bone tissue.For this purpose a number of osteoinductive carriers have been tested based on synthetic polymers, DBM, hydrogel, titanic fibres, natural coral and synthetic bioceramics based on hydroxyapatite and tricalcium phosphate (Bucholz et al. 1987;El-Ghannam 2005;Hotz and Herr 1994;Ishaug et al. 1997;Fleming et al. 2000;Hutmacher 2000;Oest et al. 2007;Srouji and Livne 2005;Wolff at al. 1994).
Synthetic materials based on hydroxyapatite and tricalcium phosphate show good ability of incorporation into bone tissue, which is due to their biocompatibility, degradability and porous structure allowing their intergrowth through newly formed bone (Bruder et al. 1998a;Marcacci et al. 1999).Their "merely" osteoinductive property, however, is insufficient for the healing of extensive bone defects (Bruder at al. 1998a).In contrast, synthetic polymers have lower ability of osetointegration compared to bioceramics, and their degradation is connected with stronger tissue reaction (Fleming 2000;Oest et al. 2007).
Attention of contemporary research is also focused on the development of threedimensional (3D) scaffolds planted with MSCs that might be used for the reconstruction of extensive bone defects.This strategy combines the osteogenic potential of MSCs with osteoconductive abilities of the scaffolds (Nečas et al. 2008;Viateau et al. 2007;Ishauga et al. 1997;Fleming et al. 2000;Kraus and Kirker-Head 2006;Kadiyala et al. 1997;Jančář et al. 2007), supplemented in some cases also with the osteopromotive component (Neuttelman et al. 2006;Kim et al. 2007;Kirker-Head et al. 2007;Rosenbaum et al. 2008).In experimental studies dealing with the reconstruction of large bone defects using 3D scaffolds planted with MSCs, the model animal used was the rat (Kadiyala et al. 1997;Bruder et al. 1998a;Srouji and Livne 2005), rabbit (Kirker-Head et al. 2007), dog (Kraus et al. 1999;Kraus and Kirker-Head 2006) and sheep (Marcacci et al. 1999;Viateau et al. 2007).In several studies, bioceramics was used as the carrier (Kraus et al. 1999;Kraus and Kirker-Head 2006).Recently published studies focus on the development and transplantation of a scaffold based on fibroin (protein derived from the silk produced by the silkworm moth caterpillar) combined with MSCs or BMPs (Kirker-Head 2007;Meinel et al. 2006), or a bioactive scaffold composed of collagen and peptide derived from osteopontin (Lee at al. 2007).
In vivo experiments on animal models yield promising results.The experimental work of Kadiyala et al. (1997) points out that transplantation of allogeneic MSCs on a hydroxyapatite/tricalcium phosphate scaffold brought about faster healing of diaphyseal femoral defects in rats than the application of BMPs with a similar scaffold.Similar results are confirmed in other studies conducted on large animal models (Kirker-Head 2006;Viateau et al. 2007).
At present, we conduct in vivo studies at our department on the transplantation of MSCs combined with mechanically resistant, biocompatible resorbable scaffolds into segmental femoral defects in miniature pigs.For fixation of these segmental defects we use LCP plates (Locking Compression Plate, Synthes) in combination with lock screws (Plate VIII, Fig. 1).The healing process is continuously evaluated using radiological examination (Plate IX, Fig. 2) and computed tomography (Plate X, Fig. 3) as well as on the basis of performed mechanical tests of the firmness of operated femurs and histological examination of defect locations.Preliminary results of this study with regard to better bone defect healing appear promising.Verification of the regenerative potential of transplanted MSCs under conditions in vivo on animal models is the first step before the presumed therapeutic application of MSCs in clinical practice.For the future, the use of the osteogenic potential of MSCs in combination with biomaterials is considered in the healing of bone lesions in humans and animals, which could substitute existing methods of bone regeneration that are in some cases insufficient.Owing to the possibility of long-term storage of MSCs and the promising results of experimental studies on allogeneic transplantation of these cells, it is possible even to consider the use of tissue banks that might operatively provide MSC cultures for clinical purposes.It will be necessary, however, to search for answers to a number of other questions related to supporting the healing of damaged bone tissue using the transplantation of stem cells combined with biomaterials.
Fig. 1.Transplantation of biocompatible scaffold seeded with MSCs into segmental femoral defect fixed with LCP plate in a miniature pig