A clinical perspective to mesenchymal stem cell-based musculoskeletal regeneration

Introduction The global increase in number in severity of traumatic and degenerative musculoskeletal diseases affects the quality of life and increases health-care costs and resource expenditure. Facing an epidemic of orthopaedic tissue pathology in all ages and economic surroundings, modern medicine is challenged to improve strategy to deliver efficient and affordable treatments. Regenerative medicine has been identified as able to offer revolutionary solutions and to enable complete structural and functional rehabilitation of tissues, organs and bodily systems. This article reviews current regenerative medicine strategies for musculoskeletal diseases of bone, tendon and cartilage, which made their way to clinical application. Stage-related disease regenerative medicine planning and phenotypic mesenchymal stem cell biomarker characterisation are identified as potential factors accelerating the clinical applicability of mesenchymal stem cell-based therapies for musculoskeletal regeneration. Conclusion Cell source profiling using biomarkers for proliferation and differentiation towards the desired lineage would be the next step in improving the regenerative medicine strategy. Introduction Regenerative medicine (RM) is sought to making possible the anatomical and functional restoration of tissues and organs impaired or lost due to disease, trauma, congenital abnormalities or advancing age1. RM uses advanced scientific knowledge in the field of molecular and developmental biology, nanomaterials, bioinformatics and computational biology to provide solutions for untreatable diseases till date, congenital abnormalities or age-related degeneration. Innovative, affordable RM solutions can help both developed and developing countries to address the increasing burden of disease costs and improving the global health equity2. In RM, cells, the ultimate ‘biological executive’ are manipulated or targeted for the structural, bioactive and/or immunomodulatory effect. Different cell sources are available and extensively studied for regenerative purposes. Mesenchymal stem cells (MSCs) are the adult stem cells involved in growth, maintenance and repair of connective tissues: cartilage, bone, tendon, and ligament and marrow stoma. MSCs are isolated based on their adherence to the plastic culture dish, and can be expanded in vitro for several passages to be used for research or clinical applications. International Society for Cell Therapy has identified the minimal criteria for the characterisation of MSCs3. Autologous MSCs of clinically significant volume can be obtained with minimal cell manipulation. In this article, the current MSC-based RM strategies for connective tissues are outlined, with a focus on bone, cartilage, tendons and ligaments repair. Discussion Bone regeneration When damaged, the bone has a remarkable capability to rebuild completely functional tissues. Nevertheless, nonunions—failure of bone healing— are challenging to treat, requiring surgery for mechanical stabilisation and/or biological augmentation. The increasing numbers of nonunions are frequently related to the increasing cases of extensive musculoskeletal injury produced by traffic accidents, war and natural calamities4. Bone loss due to trauma, tumour removal or periprosthetic loosening is a clinical situation requiring tissue substitution. For mechanical stability and osteoconductivity, natural bone grafts of various origins (autografts, allografts or xenografts) are used to treat bone defects. Autologous grafts are osteoinductive, capable to locally recruit osteoprogenitors and to induce the production of de novo mineralised tissue, therefore, considered as the golden standard in bone replacement5. However, autografts are available in limited volume expose to increased surgery time and intraoperative blood loss and can result in donor site morbidity. Osseous integration at the interface of prosthetic implants, as well as bone healing at the surgically induced fusion sites for joint or spine stabilisation are increasing the demand for bone grafts. Synthetic bone substitutes are currently used as osteoconductive replacement material. However, the poor mechanical properties and the blood supply for larger implanted volumes are limiting their clinical use. MSC-based in vivo and in vitro or ex vivo functional tissue-engineered bone grafts are regarded as a promising solution for graft shortage6. Obtaining ex vivo bone * Corresponding author Email: drlluminita@yahoo.com 1 Department of Orthopaedic Surgery, University Hospital Saint Spiridon Iasi Romania, Iasi, Romania 2 SBIM Systems Bioinformatics and Modelling GMBH, Frankfurt, Germany 3 NUI Galway Ireland NCBES National Centre for Biomedical Engineering Sciences, Galway, Ireland 4 BioTalentum, Godollo, Hungary M an ag em en t


Introduction
Regenerative medicine (RM) is sought to making possible the anatomical and functional restoration of tissues and organs impaired or lost due to disease, trauma, congenital abnormalities or advancing age 1 .RM uses advanced scientific knowledge in the field of molecular and developmental biology, nanomaterials, bioinformatics and computational biology to provide solutions for untreatable diseases till date, congenital abnormalities or age-related degeneration.Innovative, affordable RM solutions can help both developed and developing countries to address the increasing burden of disease costs and improving the global health equity 2 .In RM, cells, the ultimate 'biological executive' are manipulated or targeted for the structural, bioactive and/or immunomodulatory effect.Different cell sources are available and extensively studied for regenerative purposes.Mesenchymal stem cells (MSCs) are the adult stem cells involved in growth, maintenance and repair of connective tissues: cartilage, bone, tendon, and ligament and marrow stoma.MSCs are isolated based on their adherence to the plastic culture dish, and can be expanded in vitro for several passages to be used for research or clinical applications.International Society for Cell Therapy has identified the minimal criteria for the characterisation of MSCs 3 .Autologous MSCs of clinically significant volume can be obtained with minimal cell manipulation.In this article, the current MSC-based RM strategies for connective tissues are outlined, with a focus on bone, cartilage, tendons and ligaments repair.

Bone regeneration
When damaged, the bone has a remarkable capability to rebuild completely functional tissues.Nevertheless, nonunions-failure of bone healingare challenging to treat, requiring surgery for mechanical stabilisation and/or biological augmentation.The increasing numbers of nonunions are frequently related to the increasing cases of extensive musculoskeletal injury produced by traffic accidents, war and natural calamities 4 .Bone loss due to trauma, tumour removal or periprosthetic loosening is a clinical situation requiring tissue substitution.For mechanical stability and osteoconductivity, natural bone grafts of various origins (autografts, allografts or xenografts) are used to treat bone defects.Autologous grafts are osteoinductive, capable to locally recruit osteoprogenitors and to induce the production of de novo mineralised tissue, therefore, considered as the golden standard in bone replacement 5 .However, autografts are available in limited volume expose to increased surgery time and intraoperative blood loss and can result in donor site morbidity.Osseous integration at the interface of prosthetic implants, as well as bone healing at the surgically induced fusion sites for joint or spine stabilisation are increasing the demand for bone grafts.Synthetic bone substitutes are currently used as osteoconductive replacement material.However, the poor mechanical properties and the blood supply for larger implanted volumes are limiting their clinical use.MSC-based in vivo and in vitro or ex vivo functional tissue-engineered bone grafts are regarded as a promising solution for graft shortage 6 .Obtaining ex vivo bone grafts using MSCs seeded on scaffolds, cultivated in bioreactors with or without mechanical stimulation, proved to be less rewarding as initially thought.Some of the issues are related to the duration of the procedure, graft contamination risk, costs, scalability and product marketing approval.Moreover, large graft vascularisation remains an unsolved problem.The effort to design permeable blood vessels within the graft requires additional tissue engineering strategies, increased bioreactor time and specific implantation surgery.In vivo generation of stable functional bone appears to be a more simple procedure.It can be performed during one-step surgery; grafts can be prepared on the operating site, significantly reducing cell manipulation and contamination risk 7 .To date, autologous cell sources already used in clinical settings are bone marrow (BM) stromal cells and adipose-derived stem cell (ADSC).Unprocessed BM is particularly attractive as it can be easily obtained from the iliac crest with simple needle aspiration.Autologous BM aspirates, injected directly into the grafting site, have been used to treat atrophic nonunion 8 , lunate bone osteonecrosis 9 , delayed union or non-unions of long bones 10 .BM cellularity is donor dependent, therefore, to prevent graft paucity, cells can be enriched at the site of the surgery.A device based on the principles of the affinity column, designed to populate a scaffold with BM retained nucleated cells 11 , was used to concentrate the cells for spinal fusion site augmentation.The method was used in two clinical trials, BM concentrate combined with demineralised bone matrix induced up to 84% fusion rates and significantly improved clinical outcomes 7 .The use of MSCs in combination with three-dimensional scaffolds provides mechanical support for the cells and mechanical stability of the grafting site.Moreover, the scaffolds can be shaped to address the clinical needs; their stability can be further secured using conventional internal or external bone fixation.Results from a large variety of natural, synthetic or composite biomaterials (collagen, hydroxyapatite (HA), β tricalcium phosphate, polylactic glycolic acid (PLGA)) are available from preclinical or clinical studies 12 .BMSCs deposited on allogeneic bone graft were clinically used to treat large post-traumatic long bone defects 13 , to stabilise posterior spinal fusion, in combination with porous bone substitute, β-tri-calcium phosphate 14 .Autologous ADSCs, fibrin glue and iliac graft, induced new bone formation after 3 months in a case of extensive paediatric calvarial bone loss 15 .There are currently several clinical trials investigating autologous BMSCs or ADSCs (Table 1).Other MSC sources (umbilical cord blood and placenta) have to prove their clinical usefulness in bone regeneration.Alternative cell sources and in vitro expansion could be justified in selected cases of patients with low concentration of osteoprogenitor cells in BM aspirates.Promising results from a clinical study using threedimensional HA constructs and autologous BMMSCs reporting excellent clinical results after 6-7 years 16 are raising hopes that engineered bone could be available to the clinic in the proximate future.Preconditioning of the graft by means of dynamic bioreactor technology and methods for providing engineered bone the blood supply would be likely to accelerate this process.

Cartilage regeneration
Unlike bone, cartilage tissue has limited potential for spontaneous healing.Traumatic cartilage injuries are increasing in number and gravity of cases.Cartilage lesions as a result of sports-related injury or overuse affect mainly younger or biologically young persons who expect as complete as possible structural and functional recovery.Worldwide population ageing is also related to an increase in cartilage degenerative diseases incidence (osteoarthritis (OA) and rheumatic arthritis (RA)) and a substantial burden, in terms of quality-of-life and health expenditures.For OA and RA, joint reconstruction surgery is a successful intervention, harnessing good functional results.However, prosthetic replacement is reserved for the advanced stage of the disease, largely in the elderly.A large therapeutic gap exists in addressing the intermediary stages of degeneration and in treating younger subjects.Pharmacological and physical therapies offer limited time symptomatic improvement.RM holds the promise for biological regeneration of joint surface and sustainable function restoration.In the 1990s, a method of treating limited cartilage defects by delivery of cultivated chondrocytes, autologous cell therapy/implantation (ACT/ACI), was introduced as the first clinical application of a local cell therapy 17 19 .Patients with patellofemoral defects way up to 8.6 cm 2 improved functional score reducing pain at a 3-year followup after 'biological arthroplasty' performed with BM aspirate concentrate and type-I/III collagen matrix 20 .
A single-staged arthroscopic cartilage repair consisting of microdrilling protected by atelocollagen or fibrin gel under carbon dioxide insufflations was used in International Cartilage Repair Society grade III or IV knee defects.After 1 year, Lysholm and MOCART scores were improved and the defect filled with hyalinelike tissue 21 .Engineered MSC-based cartilage using autologous BMMSCs seeded on collagen scaffold was reported to heal clinical patellar cartilage defects and medial femoral condyle defects in OA patients in combination with high tibial osteotomy.Good filling of the defects was reported at 44 weeks after surgery as well as improved arthroscopic and histological grading score compared with cell-free control group 22 .Scaffold structure and geometry influence MSC chondrogenesis in vitro or in animal models 23 .Nanostructure scaffolds of nano-metre scale fibres, reproducing natural ECM environment size and configuration, improve MSC-based chondrogenesis and chondrocyte activity 24 .Growth factors relevant for MSC-based chondrogenesis are members of TGF-β super family, insulin growth factor, fibroblast growth factor and plateletderived growth factor, Wnt and sox-9.
TGFs isoforms activity on chondrogenesis is species dependent, TGF-β3 being more active in human MSCs than TGF-β2 25 .Wnt proteins enhance MSC chondrogenic differentiation in cross-talks with TGF-β pathway 26 .
Transcription factor sox-9 is regarded as master key regulators for chondrogenesis.Co-delivery of SOX-9 genes and anti-Cbfa-1 small-interference RNA (siRNA)-coated onto PLGA nanoparticles in human MSCs enhanced chondrogenic and inhibited osteoblast gene and protein expression 27 .Some of the particular problems with the use of MSCs for cartilage engineering are phenotypic stability and graft mineralisation.Mechanical preconditioning enhances chondrogenetic commitment 28 while hypoxic culture conditions contribute to phenotype stability and improved chondrogenesis 29 .Chondrogenic TGF-β isoform precondition 30 or anti-Cbfa-1 siRNAs prevent MSCs differentiation to hypertrophic chondrocytes and trans-differentiation to osteoblasts responsive of graft mineralisation.Autologous MSC cell therapy reduces cartilage degeneration, osteophyte remodelling, subchondral bone sclerosis and meniscal regeneration was reported in a caprine model of OA 31 .Local delivery of MSCs reduced the concentration of serum TNF-α, decreased responsiveness of T lymphocytes and synovial proliferation in a mice model of collagen-induced arthritis 32 .Single intra-articular injection of autologous MSCs prevented post-traumatic arthritis in mice 33 .Several phase 1 or 2 clinical trials are investigating the use of autologous MSCs for the treatment of cartilage diseases (Table 1).

Tendon and ligament regeneration
Tendon and ligament injuries and degenerative diseases are a widespread group of connective tissue disease affecting the adult population.There are differences in the healing capabilities of different ligaments or tendons, depending on their location (intra-or extraarticular), type of injury (partial vs. complete rupture), the degree of physiological load (lower limb vs. upper limb) and patient expectation (normal physical activity or sport professionals).This leads to a diversity of clinical situations.Some ligaments, such as knee anterior cruciate ligament (ACL), are notorious for having no tendency to heal and require surgical ligament reconstruction.Others such as knee medial collateral ligament, Achilles tendon or rotator cuff RC restore continuity by means of a scar, which can be treated conservatively depending on the rupture degree.Repair tissue has higher type-III collagen content and collagen turnover, increased total glucosaminoglycans and functionally, less tensile strength and increased elasticity.As a result, tendon-muscle unit performances decrease, consequently leading to long rehabilitation time and loss of function.Surgical reconstruction uses tendon grafts of different origin; when using allogeneic grafts, the procedure exposes to contamination risk or introduces a degree of damage in a previously intact donor zone by allograft harvest.Augmentation therapy for healing tissue and provision of tendon engineered grafts are two strategies with the potential to substantially improve quality of treatment and therapeutic decision-making for tendon and ligament disease.There are different commercially available synthetic polymers and decellularised matrix scaffolds 34 ; their use is controversial mainly due to graft stability over time and the presence of degradative products within the joint.viable tendon and ligament grafts.Several ex vivo engineering strategies proved successful in preclinical models.Type-I collagen hydrogels with non-demineralised bone at each end seeded with human BMMSCs under mechanical preconditioning produced a construct containing elongated fibroblast-like cells and ligament-like extracellular matrix 35 .Autologous MSCs seeded on microporous silk mesh exhibit fibroblast morphology 24 weeks after implantation in a pig model with ACL defect 36 .Suture augmentation promotes functional tendon and ligament regeneration.Autologous BMMSCs cultured in collagen gel deposited on the pretensioned tendon suture site, improved rabbit Achilles tendon load-related structural and material properties 37 .BMMSCs cultured under hypoxic condition improved collagen content and ultimate failure load in rats Achilles tendon suture site 38 .Biological augmentation uses local growth factors delivery to promote one or several stages of wound healing.Autologous MSCs genetically modified by adenovirus mediated in vitro BMP-12 gene transfer in a chicken model of tendon laceration resulted in increased repair biomechanics 39 .Plasmid transfected MSC cell lines to overexpress Smad8 and BMP-2 were able to produce neo-tendon tissue when ectopically implanted in mice 40 .One phase II clinical trial investigates the safety and efficiency of autologous MSCs on a collagen membrane for augmentation of rotator cuff repair (Table 1).

Clinical applicability of musculoskeletal RM. moving forward
RM introduces new possibilities for improving existing therapies and answering unmet clinical needs in the field of musculoskeletal disease.A practical approach to advancing science and facilitating clinical application would be a more sustained effort in improving collaboration between different experts implicated in the design of regenerative approaches for a specific indication.The earlier in the basic research phase this dialogue intervenes; the future product is more likely to target a clinical situation and to obtain larger applicability and improved performances.A 'bottom-top' approach, starting the basic research effort from clinical necessities identified by panels of clinicians, biologists and bioengineers could more precisely delineate directions for RM development.Different stages of the same disease are characterised by particular normal and abnormal molecular pathways and by distinct mechanical situation at macro and ultra-structural level.Better tailored RM strategies will need to target more than a particular disease but also biological and biomechanical particularities, which characterise pre-defined pathological stages.To this end, a molecular classification of musculoskeletal diseases would be highly desirable, with the capability to discriminate the intimate structural and functional changes, offering targets for regenerative strategies.The already well-known triad cellsscaffolds-bioactive molecules could be used selectively, in part or in various combinations to better fit the clinical situation.

Conclusion
Cell source profiling using biomarkers for proliferation and differentiation toward the desired lineage would be the next step in improving the RM strategy.Algorithm-based identification of stage of the disease, profiling of donor and host biology as well as in silico models of cell engraftment after implantation will help in characterising RM product.Flexible, interchangeable pipelines where patient data could be incorporated in early stages of production would result in personalised and affordable regenerative therapies.
. Matrix-assisted chondrocyte implantation/transplantation (MACI/MACT) addresses the problem of cell retention within the defect, in the mean time allowing for phenotype preservation in a threedimensional (3D) environment.These technique limitations are mainly related to the biological potential of differentiated cells.Healthy cartilage biopsy can derive only limited amounts of cells; moreover, their expansion capability decreases with age and associated diseases.Cartilage donor site can become symptomatic and be the source of joint degradation.For reasons of larger availability, proliferation and differentiation potential, the use of MSCs as the cell source is sought to facilitate cartilage engineering.Threedimensional high-density pellet or micromass-cultured MSCs exposed to specific differentiation media and growth factors (transforming growth factor (TGF)-β1, 2 and 3, bone morphogenetic protein (BMP)-2, 4 or 6, IGF-1) are used in vitro to obtain functional, type-I collagen and glycosaminoglycan producing chondrocytes.Similarly, MSCs seeded on various scaffolds exposed to chondrogenetic media are shown to Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) FOR CITATION PURPOSES: Labusca L, O'Brien M, Mashayekhi K.A clinical perspective to mesenchymal stem cell-based musculoskeletal regeneration.OA Musculoskeletal Medicine 2013 May 01;1(1):8.
Autologous MSC-based tendon engineering is regarded as an appealing perspective to producing long lasting, Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) FOR CITATION PURPOSES: Labusca L, O'Brien M, Mashayekhi K.A clinical perspective to mesenchymal stem cell-based musculoskeletal regeneration.OA Musculoskeletal Medicine 2013 May 01;1(1):8.

Table 1 . Clinical trials involving MSC of diff erent sources for bone and carti lage repair a,b . Tissue Number Cell source Cell culture Method Intenti on to treat Study Country Study ID
18, bone engineering; CE, cartilage engineering; TE, tendon engineering; NU, non union; DU, delayed union.ahttp://www.clinicaltrials.gov/ct2/results?term=mesenchymal+stem+cells+bone+repair&pg=2 (accessed 14 May 2013).bhttp://www.clinicaltrials.gov/ct2/results?term=cartilage+repair+stem+cells+&Search=Search (accessed 23 May 2013); http://www.clinicaltrials.gov/ct2/show/NCT01687777?term=tendon+AND+repair+AND+stem+cells&rank=1 (accessed 25 May 2013).Licensee OA Publishing London 2013.Creative Commons Attribution Licence (CC-BY) FOR CITATION PURPOSES: Labusca L, O'Brien M, Mashayekhi K.A clinical perspective to mesenchymal stem cell-based musculoskeletal regeneration.OA Musculoskeletal Medicine 2013 May 01;1(1):8.assumeachondrocyte-likephenotype, making the design of implantable grafts achievable.Reports about various strategies of grafting defects with engineered cartilage in animal models are abundant18.To date, the existent RM clinical application reported can be grouped into protected autologous bone marrow stimulation techniques, MSC-based engineered cartilage engineering and cell therapy.The first is based on using a method of subchondral bone stimulation (microfracture) in combination with a sealant of the cartilage defect (e.g. a glued type-I, II and III collagen membrane).For clinically limited knee cartilage defects, Autologous Matrix Induced Chondrogenesis, AMIC ® is reported reducing pain and improving Lysholm score in a medium follow up