Osteoarthritis and Cartilage 22 (2014) 2013e2016
Review
Osteoarthritis Year in Review 2014: highlighting innovations in basic research and clinical applications in regenerative medicine G.J.V.M. van Osch* Department of Orthopaedics and Department of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, The Netherlands
a r t i c l e i n f o
s u m m a r y
Article history: Received 12 May 2014 Accepted 22 July 2014
Regenerative medicine is an emerging area that will influence the treatment of joint diseases in the future. It involves the use of biomaterials, cell therapy, and bioactive factors such as growth factors, drugs and small molecules, to regenerate damaged tissues. This “year in review” highlights a personal selection of promising studies published between March 2013 and March 2014 that inform on the direction in which this field is moving. This multidisciplinary field has been very active, with rapid development of new technologies that emerge from basic sciences such as the possibility to generate pluripotent stem cells without genetic modification and genetic engineering of growth factors to enhance their capacity to induce tissue repair. The increasing knowledge of the interaction between all tissues in the joint, such as the effect of bone remodeling and synovial inflammation on cartilage repair, will eventually make tissue regeneration in a compromised joint environment possible. © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Regenerative medicine Cartilage Biomaterials Stem cells Growth factors
Introduction Approximately 25 years ago, three separate papers were published that kicked off the field of musculoskeletal regenerative medicine: Langer and Vacanti published the first definition of Tissue Engineering1; cells isolated from the bone marrow were demonstrated to have multilineage differentiation potential2; and the first cell transplantations for articular cartilage defects were performed in humans3. Many papers are being published in the field of stem cell biology, on the application of growth factors and on the development and application of biomaterials. A search in PubMed (ncbi.nlm.gov/pubmed/) revealed over 6000 hits in the past year on regeneration/repair of joint tissues (excluding bone there are still over 2000 publications) and over 25 new clinical trials on joint regeneration therapies were registered (clinicaltrials. gov). This review is a personal selection of articles published between March 2013 and March 2014 in the field of regenerative medicine which results are or might become important for the development of treatments to regenerate damaged joint tissues.
* Address correspondence and reprint requests to: G.J.V.M. van Osch, Erasmus MC, University Medical Center Rotterdam, Connective Tissue Cells and Repair Group, Department Orthopaedics & Department Otorhinolaryngology, Room Ee1655, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. Tel: 31-10-7043661. E-mail address: [email protected].
This review is not meant as a complete overview of publications of the year. Advances in stem cell biology To regenerate tissue differentiated cells would be the first choice. However, this will require harvesting of tissue thus creating a tissue defect and, moreover, the capacity to expand these cells in order to obtain a sufficient number for clinical applications, is limited. Mesenchymal Stem Cells (MSCs) from adult tissues are often the next choice. They are generally obtained from patient's own bone marrow or adipose tissue but other sources, such as the umbilical cord, are becoming popular as well. The MSCs however, are a very heterogeneous population of cells and the variability between donors is high4,5. In 2012 the Nobel prize for medicine was awarded to Shinya Yamanaka for the work on induced pluripotent stem cells. Yamanaka and colleagues described a way to reprogram adult somatic cells to pluripotent stem cells by genetically introducing a set of transcription factors first in murine cells (2006)6 and shortly after in human cells (2007)7. Since then the technology became more wide spread and its efficiency has increased. Apart from their use in patient-characteristic in vitro models the interest to use these cells for regenerative medicine applications in patients has grown. This year two papers were published on the attempt to eliminate the requirement of genetic modification to induce pluripotency. In the first paper Hou et al.8 described the possibility
http://dx.doi.org/10.1016/j.joca.2014.07.022 1063-4584/© 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
2014
G.J.V.M. van Osch / Osteoarthritis and Cartilage 22 (2014) 2013e2016
to induce pluripotency with a combination of four small molecules: CHIR, a glycogen synthase kinase 3 inhibitor; 616452, a Transforming Growth Factor-beta inhibitor; Forskolin, a cAMP agonist and DZNep, an S-adenosyl homocyteine hydrolase inhibitor, essential for Chemically induced Pluripotent Stem Cells (CiPSCs). In the second paper, Obokata et al.9 published a very intriguing set of data on the reprogramming of cells by exposure to sub-lethal environmental cues. Exposure of CD45þ cells from spleens of 1 week old mice to pH 5.4e5.8 for 30 min followed by 7 days culture, resulted in expression of Oct4 in the surviving cells, indicating cell reprogramming. These cells obtained by Stimulus-Triggered Acquisition of Pluripotency (STAP) showed multilineage differentiation potential and the potency to form teratomas and chimaeric mice. When cultured in medium with Leukemia IFactor, B27 and Adrencorticotropic hormone (ACTH), STAP cells have the potential to proliferate and acquire stem cell-like properties. A few other physical stimuli also gave this effect such as trituration through capillary pipettes for 20 min and application of streptolysine for 2 h to damage the cell membrane. Many groups all over the world are repeating these experiments to confirm the findings, demonstrating the high interest in this technology. Many critical notes were made in the months following the publication and in July the authors have retracted the paper. Multiple errors were found that impair the credibility of the study as a whole. The authors stated that they are unable to say without doubt whether the STAP stem cells are real. Ongoing studies in the lead authors' institute should reveal this and if the data can be replicated the potency of this method as well as the possible application in adult human cells should become clear afterwards. One of the main drawbacks of the use of pluripotent cells for application in patients is the risk of unwanted tissue formation. Transdifferentiation of cells from one lineage directly into another lineage without reverting to the pluripotency state might be a solution. Adult human fibroblasts were transdifferentiated into chondrocytes by viral transduction with two reprogramming factors, c-Myc and Kruppel-like factor 4 (Klf4), and a chondrogenic factor, Sox910. These cells produce cartilage without going through the pluripotent state and they did not form tumors. However, these cells had only very limited proliferative capacity. The field is still searching for the best cell sources for each regenerative medicine applications thereby continuously weighing the importance of cell availability and donor site morbidity, cell expansion possibilities and costs and the ability, stability and safety of the cells to perform the required function. Advances in the use of growth factors Growth factors are known for their tissue repair activities. However, high concentrations are required for application of growth factors in vivo which causes safety issues and high costs. Slow release systems and gene transfer techniques to increase production of growth factors by cells are being applied frequently in the lab to circumvent these problems. To improve the function of growth factors, genetic engineering can be used. Last year Martino.et al.11 engineered growth factors that bind better to the extra-cellular matrix and thus are active for longer period of time9. They first investigated the ability of a large number of growth factors to bind to matrix proteins. This demonstrated that Placenta Growth Factors 2 (PIGF2) had a high affinity for many matrix proteins. Its splice variant PIGF1, which does not have the heparinbinding sequence that PIGF2 has, had a much lower binding affinity. By genetically fusing the PlGF2 heparin-binding domain with other commonly used growth factors such as Bone Morphogenetic Protein 2 (BMP2), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), they showed increased skin
and bone repair. This was not the only example of engineering growth factors with improved activity. Introducing BMP2 derived residues into the Bone Morphogenetic Protein Receptor 1 (BMPR1) binding region of Growth Differentiation Factor 5 (GDF5) resulted in a 10-fold increase in binding of GDF5 to BMPR1a thereby increasing its bone forming capacity12. Since the modification was very small, the angiogenic properties of GDF5 were unaltered thus combining the positive effects of BMP2 and GDF5 in the newly engineered growth factor resulting in superior bone formation. Of great interest for regenerative medicine applications in the clinical setting is the possible use of Platelet Rich Plasma (PRP), which contains high concentrations of endogenous growth factors. The product can easily be made from patient own blood and then applied with low risk. Initially PRP was shown to stimulate cell proliferation and extra-cellular matrix production, and now an increasing number of studies indicate its anti-inflammatory effects13,14. The high number of reviews written on PRP in the last year indicates its popularity. But although the clinical studies performed so far show promising results, there is still limited evidence for efficacy because of the lack of high quality studies15. Advances in the use of biomaterials Many synthetic and natural materials have been developed and evaluated as scaffold or carrier of cells and bioactive factors to stimulate tissue repair. One approach that has become very popular and appealing for tissue repair is the use of decellularised tissues. The principle of this technology is to remove the cells and therefore the antigenicity of the tissue whereas retaining the structural functionality of the extracellular matrix. The technology has attention for whole organ replacements such as liver, heart and lung. The first clinical application of decellularised tissue in patients was actually done with cartilage to successfully replace part of the airway16. Since then protocols for various types of cartilage are being optimized to obtain good cell removal but at the same time preserve as good as possible the structural and mechanical properties of the tissue. Besides their direct application in surgical reconstructions, these materials, based on natural, bioactive components, can be expected be applied in biotechnological procedures to obtain cells or tissues for tissue engineering procedures. At the moment cell seeding in these materials appears to be a major challenge. Inspired by the decellularisation approach, people have started to produce cell culture derived extracellular matrices in the lab that can be decellularised to generate a cell-free materials17. These materials can then be used as scaffold to study the effect of cellspecific extracellular matrix on cellular behavior or to in future possibly also to seed autologous cells for tissue reconstructions. A very nice example of translational research from development in the lab towards a level 1 clinical study is BTS-CarGel18. This mixture of chitosan gel with autologous blood, was investigated for the treatment of cartilage defects in a multicenter randomized controlled study involving 26 centers from all over the world. The study included 39 patients in the BTS-CarGel treatment group and 41 that were treated with conventional microfracture. The primary outcome measure was blinded evaluation of 3D defect filling and T2 relaxation on Magnetic Resonance Imaging (MRI) after 12 months. This demonstrated statistically significantly more lesion filling and more hyaline cartilage in the BST-CarGel group. Although there were no difference in clinical improvement of the patients and it remains to be seen in future whether the benefit on the long term is good enough, the method demonstrated to be safe, affordable and easy to apply. This method was optimized from a laboratory setting and basic research is still being performed to further understand the process,
G.J.V.M. van Osch / Osteoarthritis and Cartilage 22 (2014) 2013e2016
possibly leading to further optimization. In a study with adult rabbits, hyaline cartilage regeneration after microdrilled holes with chitosan/blood implant was associated with increased remodeling of the bone19. Bone remodeling processes can affect cartilage, as we know from osteoarthritis. High levels of TGF-beta (that could be released either from the macrophages, osteoclasts or from the bone matrix) can stimulate bone resorption by osteoclasts, as well as angiogenesis, bone formation by MSCs and chondrogenesis. Blocking TGF-beta signaling in nestin-positive MSCs in bone was shown to prevent changes in bone architecture and reduce osteoarthritis development after transection of the anterior cruciate ligament in mice and rats20. So increased subchondral bone remodeling can be either positive17 or negative20 for the cartilage. This confirms the importance of understanding the crosstalk between the bone-cartilage unit. To facilitate studies on the role of subchondral bone in cartilage repair, an in vitro defect model in young bovine osteochondral biopsies was developed that showed that factors secreted by living bone can induce chondrogenic differentiation in MSCs21. Knowledge on the influence of bone on cartilage regeneration, the role of growth factors and cells and the effect of compromised bone in joint diseases is of importance to improve the treatment of cartilage lesions. Regeneration and (joint) inflammation Whenever we need tissue regeneration, we are dealing with a compromised environment that will have signs of inflammatory processes. Joint inflammation is generally believed to have adverse effects on cartilage repair. Using mice of different genetic backgrounds, an inverse relationship was shown between cartilage healing and susceptibility for posttraumatic osteoarthritis22. Superhealer Murphy Roths Large (MRL/MpJ) mice that have demonstrated the capacity to heal articular cartilage23, appeared to have less inflammation than C57Bl6 mice after intra-articular fracture and their fractures indeed resulted less frequently in posttraumatic arthritis24. In another study the same group investigated whether MSCs of MRL mice have better regenerative capacity than the MSCs of C57Bl6 mice25. Bone marrow derived MSCs from MRL/MpJ mice appeared to form more colonies but did not have a better differentiation capacity, which might indicate better regenerative capacity. Moreover, MSCs from both mouse strains injected after intra-articular fracture in the knee of the badly healing C57Bl6 mice, appeared to prevent the development of posttraumatic arthritis25. Presumably, the injected MSCs caused a mild increase in anti-inflammatory cytokine levels in the synovial fluid. This anti-inflammatory effect of MSCs on joint tissues has been shown in vitro with human bone marrow derived MSCs26 as well as human adipose derived MSCs27, making stem cells still a promising cell source for the treatment of osteoarthritis. The number of clinical studies using stem cells beyond the stage of case series is increasing and dose response safety studies have been conducted28. However, inflammation is not only preventing tissue regeneration. Attraction of more macrophages by the recruitment agent SEW2871 and PRP hydrogel, lead to more bone regeneration in rats29. A crucial role for macrophages in tissue regeneration was also demonstrated in salamanders30. Early after amputation of the salamander limb, anti-inflammatory macrophages appear along with the pro-inflammatory macrophages. Removal of macrophages using clodronate liposomes prevented limb regeneration. More knowledge about the dual role of inflammation in tissue regeneration is required. Targeting the timing of macrophage presence and macrophage phenotype in mammalian wound healing processes will probably lead to enhanced outcome of tissue repair opening new possibilities in the regeneration of tissues in inflamed environment such as an osteoarthritic joint.
2015
Concluding remarks Fast progression in the fields of stem cell biology, growth factors and biomaterials and the improving multidisciplinary relations between biologists, pathologists, immunologists, engineers and clinical scientists may in the future offer possibilities to regenerate joint tissues when prevention of osteoarthritis has failed.
Author contribution Gerjo van Osch searched the literature, selected the articles, summarized the results and wrote the manuscript.
Conflict of interest The author has no conflicts of interest.
References 1. Langer R, Vacanti JP. Tissue engineering. Science 1993;260: 920e6. 2. Pittenger MF, Mackay AM, Beck SC, Jaiswal RK, Douglas R, Mosca JD, et al. Multilineage potential of adult human mesenchymal stem cells. Science 1999;284:143e7. 3. Brittberg M, Lindahl A, Nilsson A, Ohlsson C, Isaksson O, Peterson L. Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. N Engl J Med 1994;331:889e95. 4. Muraglia A, Cancedda R, Quarto R. Clonal mesenchymal progenitors from human bone marrow differentiate in vitro according to a hierarchical model. J Cell Sci 2000 Apr;113(Pt 7): 1161e6. 5. Siddappa R, Licht R, van Blitterswijk C, de Boer J. Donor variation and loss of multipotency during in vitro expansion of human mesenchymal stem cells for bone tissue engineering. J Orthop Res 2007 Aug;25(8):1029e41. 6. Takahashi K, Yamanaka S. Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 2006;126:663e76. 7. Takahashi K, Tanabe K, Ohnuki M, Narita M, Ichisaka T, Tomoda K, et al. Induction of pluripotent stem cells from adult human fibroblasts by defined factors. Cell 2007;131:861e72. 8. Hou P, Li Y, Zhang X, Liu C, Guan J, Li H, et al. Pluripotent stem cells induced from mouse somatic cells by small-molecule compounds. Science 2013;341:651e4. 9. Obokata H, Wakayama T, Sasai Y, Kojima K, Vacanti MP, Niwa H, et al. Stimulus-triggered fate conversion of somatic cells into pluripotency. Nature 2014;505:641e7. 10. Outani H, Okada M, Yamashita A, Nakagawa K, Yoshikawa H, Tsumaki N. Direct induction of chondrogenic cells from human dermal fibroblast culture by defined factors. PLoS One 2013;8: e77365. 11. Martino MM, Briquez PS, Güç E, Tortelli F, Kilarski WW, Metzger S, et al. Growth factors engineered for super-affinity to the extracellular matrix enhance tissue healing. Science 2014;343:885e8. 12. Kleinschmidt K, Ploeger F, Nickel J, Glockenmeier J, Kunz P, Richter W. Enhanced reconstruction of long bone architecture by a growth factor mutant combining positive features of GDF5 and BMP-2. Biomaterials 2013;34:5926e36. 13. Sundman EA, Cole BJ, Karas V, Della Valle C, Tetreault MW, Mohammed HO, et al. The anti-inflammatory and matrix restorative mechanisms of platelet-rich plasma in osteoarthritis. Am J Sports Med 2014;42(1):35e41.
2016
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14. Andia I, Maffulli N. Platelet-rich plasma for managing pain and inflammation in osteoarthritis. Nat Rev Rheumatol 2013;9(12):721e30. 15. Chang KV, Hung CY, Aliwarga F, Wang TG, Han DS, Chen WS. Comparative effectiveness of platelet-rich plasma injections for treating knee joint cartilage degenerative pathology: a systematic review and meta-analysis. Arch Phys Med Rehabil 2014;95:562e75. 16. Macchiarini P, Jungebluth P, Go T, Asnaghi MA, Rees LE, Cogan TA, et al. Clinical transplantation of a tissue-engineered airway. Lancet 2008;372:2023e30. 17. Tightly anchored tissue-mimetic matrices as instructive stem cell microenvironments. Nat Methods 2013;10:788e94. 18. Stanish WD, McCormack R, Forriol F, Mohtadi N, Pelet S, Desnoyers J, et al. Novel scaffold-based BST-CarGel treatment results in superior cartilage repair compared with microfracture in a randomized controlled trial. J Bone Joint Surg Am 2013;95:1640e50. n-Morales J, Lafantaisie-Favreau CH, Chen G, 19. Guzma Hoemann CD. Subchondral chitosan/blood implant-guided bone plate resorption and woven bone repair is coupled to hyaline cartilage regeneration from microdrill holes in aged rabbit knees. Osteoarthritis Cartilage 2014;22:323e33. 20. Zhen G, Wen C, Jia X, Li Y, Crane JL, Mears SC et al. Inhibition of TGF-b signaling in mesenchymal stem cells of subchondral bone attenuates osteoarthritis. Nat Med;19:704e12. 21. de Vries-van Melle ML, Narcisi R, Kops N, Koevoet WJ, Bos PK, Murphy JM, et al. Chondrogenesis of mesenchymal stem cells in an osteochondral environment is mediated by the subchondral bone. Tissue Eng Part A 2014;20:23e33. 22. Ward BD, Furman BD, Huebner JL, Kraus VB, Guilak F, Olson SA. Absence of posttraumatic arthritis following intraarticular fracture in the MRL/MpJ mouse. Arthritis Rheum 2008;58: 744e53.
23. Fitzgerald J, Rich C, Burkhardt D, Allen J, Herzka AS, Little CB. Evidence for articular cartilage regeneration in MRL/MpJ mice. Osteoarthritis Cartilage 2008;16:1319e26. 24. Lewis Jr JS, Furman BD, Zeitler E, Huebner JL, Kraus VB, Guilak F, et al. Genetic and cellular evidence of decreased inflammation associated with reduced incidence of posttraumatic arthritis in MRL/MpJ mice. Arthritis Rheum 2013;65: 660e70. 25. Diekman BO, Wu CL, Louer CR, Furman BD, Huebner JL, Kraus VB, et al. Intra-articular delivery of purified mesenchymal stem cells from C57BL/6 or MRL/MpJ superhealer mice prevents posttraumatic arthritis. Cell Transplant 2013;22: 1395e408. 26. van Buul GM, Villafuertes E, Bos PK, Waarsing JH, Kops N, Narcisi R, et al. Mesenchymal stem cells secrete factors that inhibit inflammatory processes in short-term osteoarthritic synovium and cartilage explant culture. Osteoarthritis Cartilage 2012;20(10):1186e96. 27. Manferdini C, Maumus M, Gabusi E, Piacentini A, Filardo G, Peyrafitte JA, et al. Adipose-derived mesenchymal stem cells exert antiinflammatory effects on chondrocytes and synoviocytes from osteoarthritis patients through prostaglandin E2. Arthritis Rheum 2013;65(5):1271e81. 28. Jo CH, Lee YG, Shin WH, Kim H, Chai JW, Jeong EC, et al. Intraarticular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial. Stem Cells 2014;32(5):1254e66. 29. Kim YH, Furuya H, Tabata Y. Enhancement of bone regeneration by dual release of a macrophage recruitment agent and platelet-rich plasma from gelatin hydrogels. Biomaterials 2014;35:214e24. 30. Godwin JW, Pinto AR, Rosenthal NA. Macrophages are required for adult salamander limb regeneration. Proc Natl Acad Sci USA 2013;110:9415e20.
Review
Osteoarthritis Year in Review 2014: highlighting innovations in basic research and clinical applications in regenerative medicine G.J.V.M. van Osch* Department of Orthopaedics and Department of Otorhinolaryngology, Erasmus MC, University Medical Center Rotterdam, The Netherlands
a r t i c l e i n f o
s u m m a r y
Article history: Received 12 May 2014 Accepted 22 July 2014
Regenerative medicine is an emerging area that will influence the treatment of joint diseases in the future. It involves the use of biomaterials, cell therapy, and bioactive factors such as growth factors, drugs and small molecules, to regenerate damaged tissues. This “year in review” highlights a personal selection of promising studies published between March 2013 and March 2014 that inform on the direction in which this field is moving. This multidisciplinary field has been very active, with rapid development of new technologies that emerge from basic sciences such as the possibility to generate pluripotent stem cells without genetic modification and genetic engineering of growth factors to enhance their capacity to induce tissue repair. The increasing knowledge of the interaction between all tissues in the joint, such as the effect of bone remodeling and synovial inflammation on cartilage repair, will eventually make tissue regeneration in a compromised joint environment possible. © 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
Keywords: Regenerative medicine Cartilage Biomaterials Stem cells Growth factors
Introduction Approximately 25 years ago, three separate papers were published that kicked off the field of musculoskeletal regenerative medicine: Langer and Vacanti published the first definition of Tissue Engineering1; cells isolated from the bone marrow were demonstrated to have multilineage differentiation potential2; and the first cell transplantations for articular cartilage defects were performed in humans3. Many papers are being published in the field of stem cell biology, on the application of growth factors and on the development and application of biomaterials. A search in PubMed (ncbi.nlm.gov/pubmed/) revealed over 6000 hits in the past year on regeneration/repair of joint tissues (excluding bone there are still over 2000 publications) and over 25 new clinical trials on joint regeneration therapies were registered (clinicaltrials. gov). This review is a personal selection of articles published between March 2013 and March 2014 in the field of regenerative medicine which results are or might become important for the development of treatments to regenerate damaged joint tissues.
* Address correspondence and reprint requests to: G.J.V.M. van Osch, Erasmus MC, University Medical Center Rotterdam, Connective Tissue Cells and Repair Group, Department Orthopaedics & Department Otorhinolaryngology, Room Ee1655, Wytemaweg 80, 3015 CN Rotterdam, The Netherlands. Tel: 31-10-7043661. E-mail address: [email protected].
This review is not meant as a complete overview of publications of the year. Advances in stem cell biology To regenerate tissue differentiated cells would be the first choice. However, this will require harvesting of tissue thus creating a tissue defect and, moreover, the capacity to expand these cells in order to obtain a sufficient number for clinical applications, is limited. Mesenchymal Stem Cells (MSCs) from adult tissues are often the next choice. They are generally obtained from patient's own bone marrow or adipose tissue but other sources, such as the umbilical cord, are becoming popular as well. The MSCs however, are a very heterogeneous population of cells and the variability between donors is high4,5. In 2012 the Nobel prize for medicine was awarded to Shinya Yamanaka for the work on induced pluripotent stem cells. Yamanaka and colleagues described a way to reprogram adult somatic cells to pluripotent stem cells by genetically introducing a set of transcription factors first in murine cells (2006)6 and shortly after in human cells (2007)7. Since then the technology became more wide spread and its efficiency has increased. Apart from their use in patient-characteristic in vitro models the interest to use these cells for regenerative medicine applications in patients has grown. This year two papers were published on the attempt to eliminate the requirement of genetic modification to induce pluripotency. In the first paper Hou et al.8 described the possibility
http://dx.doi.org/10.1016/j.joca.2014.07.022 1063-4584/© 2014 Osteoarthritis Research Society International. Published by Elsevier Ltd. All rights reserved.
2014
G.J.V.M. van Osch / Osteoarthritis and Cartilage 22 (2014) 2013e2016
to induce pluripotency with a combination of four small molecules: CHIR, a glycogen synthase kinase 3 inhibitor; 616452, a Transforming Growth Factor-beta inhibitor; Forskolin, a cAMP agonist and DZNep, an S-adenosyl homocyteine hydrolase inhibitor, essential for Chemically induced Pluripotent Stem Cells (CiPSCs). In the second paper, Obokata et al.9 published a very intriguing set of data on the reprogramming of cells by exposure to sub-lethal environmental cues. Exposure of CD45þ cells from spleens of 1 week old mice to pH 5.4e5.8 for 30 min followed by 7 days culture, resulted in expression of Oct4 in the surviving cells, indicating cell reprogramming. These cells obtained by Stimulus-Triggered Acquisition of Pluripotency (STAP) showed multilineage differentiation potential and the potency to form teratomas and chimaeric mice. When cultured in medium with Leukemia IFactor, B27 and Adrencorticotropic hormone (ACTH), STAP cells have the potential to proliferate and acquire stem cell-like properties. A few other physical stimuli also gave this effect such as trituration through capillary pipettes for 20 min and application of streptolysine for 2 h to damage the cell membrane. Many groups all over the world are repeating these experiments to confirm the findings, demonstrating the high interest in this technology. Many critical notes were made in the months following the publication and in July the authors have retracted the paper. Multiple errors were found that impair the credibility of the study as a whole. The authors stated that they are unable to say without doubt whether the STAP stem cells are real. Ongoing studies in the lead authors' institute should reveal this and if the data can be replicated the potency of this method as well as the possible application in adult human cells should become clear afterwards. One of the main drawbacks of the use of pluripotent cells for application in patients is the risk of unwanted tissue formation. Transdifferentiation of cells from one lineage directly into another lineage without reverting to the pluripotency state might be a solution. Adult human fibroblasts were transdifferentiated into chondrocytes by viral transduction with two reprogramming factors, c-Myc and Kruppel-like factor 4 (Klf4), and a chondrogenic factor, Sox910. These cells produce cartilage without going through the pluripotent state and they did not form tumors. However, these cells had only very limited proliferative capacity. The field is still searching for the best cell sources for each regenerative medicine applications thereby continuously weighing the importance of cell availability and donor site morbidity, cell expansion possibilities and costs and the ability, stability and safety of the cells to perform the required function. Advances in the use of growth factors Growth factors are known for their tissue repair activities. However, high concentrations are required for application of growth factors in vivo which causes safety issues and high costs. Slow release systems and gene transfer techniques to increase production of growth factors by cells are being applied frequently in the lab to circumvent these problems. To improve the function of growth factors, genetic engineering can be used. Last year Martino.et al.11 engineered growth factors that bind better to the extra-cellular matrix and thus are active for longer period of time9. They first investigated the ability of a large number of growth factors to bind to matrix proteins. This demonstrated that Placenta Growth Factors 2 (PIGF2) had a high affinity for many matrix proteins. Its splice variant PIGF1, which does not have the heparinbinding sequence that PIGF2 has, had a much lower binding affinity. By genetically fusing the PlGF2 heparin-binding domain with other commonly used growth factors such as Bone Morphogenetic Protein 2 (BMP2), Vascular Endothelial Growth Factor (VEGF) and Platelet Derived Growth Factor (PDGF), they showed increased skin
and bone repair. This was not the only example of engineering growth factors with improved activity. Introducing BMP2 derived residues into the Bone Morphogenetic Protein Receptor 1 (BMPR1) binding region of Growth Differentiation Factor 5 (GDF5) resulted in a 10-fold increase in binding of GDF5 to BMPR1a thereby increasing its bone forming capacity12. Since the modification was very small, the angiogenic properties of GDF5 were unaltered thus combining the positive effects of BMP2 and GDF5 in the newly engineered growth factor resulting in superior bone formation. Of great interest for regenerative medicine applications in the clinical setting is the possible use of Platelet Rich Plasma (PRP), which contains high concentrations of endogenous growth factors. The product can easily be made from patient own blood and then applied with low risk. Initially PRP was shown to stimulate cell proliferation and extra-cellular matrix production, and now an increasing number of studies indicate its anti-inflammatory effects13,14. The high number of reviews written on PRP in the last year indicates its popularity. But although the clinical studies performed so far show promising results, there is still limited evidence for efficacy because of the lack of high quality studies15. Advances in the use of biomaterials Many synthetic and natural materials have been developed and evaluated as scaffold or carrier of cells and bioactive factors to stimulate tissue repair. One approach that has become very popular and appealing for tissue repair is the use of decellularised tissues. The principle of this technology is to remove the cells and therefore the antigenicity of the tissue whereas retaining the structural functionality of the extracellular matrix. The technology has attention for whole organ replacements such as liver, heart and lung. The first clinical application of decellularised tissue in patients was actually done with cartilage to successfully replace part of the airway16. Since then protocols for various types of cartilage are being optimized to obtain good cell removal but at the same time preserve as good as possible the structural and mechanical properties of the tissue. Besides their direct application in surgical reconstructions, these materials, based on natural, bioactive components, can be expected be applied in biotechnological procedures to obtain cells or tissues for tissue engineering procedures. At the moment cell seeding in these materials appears to be a major challenge. Inspired by the decellularisation approach, people have started to produce cell culture derived extracellular matrices in the lab that can be decellularised to generate a cell-free materials17. These materials can then be used as scaffold to study the effect of cellspecific extracellular matrix on cellular behavior or to in future possibly also to seed autologous cells for tissue reconstructions. A very nice example of translational research from development in the lab towards a level 1 clinical study is BTS-CarGel18. This mixture of chitosan gel with autologous blood, was investigated for the treatment of cartilage defects in a multicenter randomized controlled study involving 26 centers from all over the world. The study included 39 patients in the BTS-CarGel treatment group and 41 that were treated with conventional microfracture. The primary outcome measure was blinded evaluation of 3D defect filling and T2 relaxation on Magnetic Resonance Imaging (MRI) after 12 months. This demonstrated statistically significantly more lesion filling and more hyaline cartilage in the BST-CarGel group. Although there were no difference in clinical improvement of the patients and it remains to be seen in future whether the benefit on the long term is good enough, the method demonstrated to be safe, affordable and easy to apply. This method was optimized from a laboratory setting and basic research is still being performed to further understand the process,
G.J.V.M. van Osch / Osteoarthritis and Cartilage 22 (2014) 2013e2016
possibly leading to further optimization. In a study with adult rabbits, hyaline cartilage regeneration after microdrilled holes with chitosan/blood implant was associated with increased remodeling of the bone19. Bone remodeling processes can affect cartilage, as we know from osteoarthritis. High levels of TGF-beta (that could be released either from the macrophages, osteoclasts or from the bone matrix) can stimulate bone resorption by osteoclasts, as well as angiogenesis, bone formation by MSCs and chondrogenesis. Blocking TGF-beta signaling in nestin-positive MSCs in bone was shown to prevent changes in bone architecture and reduce osteoarthritis development after transection of the anterior cruciate ligament in mice and rats20. So increased subchondral bone remodeling can be either positive17 or negative20 for the cartilage. This confirms the importance of understanding the crosstalk between the bone-cartilage unit. To facilitate studies on the role of subchondral bone in cartilage repair, an in vitro defect model in young bovine osteochondral biopsies was developed that showed that factors secreted by living bone can induce chondrogenic differentiation in MSCs21. Knowledge on the influence of bone on cartilage regeneration, the role of growth factors and cells and the effect of compromised bone in joint diseases is of importance to improve the treatment of cartilage lesions. Regeneration and (joint) inflammation Whenever we need tissue regeneration, we are dealing with a compromised environment that will have signs of inflammatory processes. Joint inflammation is generally believed to have adverse effects on cartilage repair. Using mice of different genetic backgrounds, an inverse relationship was shown between cartilage healing and susceptibility for posttraumatic osteoarthritis22. Superhealer Murphy Roths Large (MRL/MpJ) mice that have demonstrated the capacity to heal articular cartilage23, appeared to have less inflammation than C57Bl6 mice after intra-articular fracture and their fractures indeed resulted less frequently in posttraumatic arthritis24. In another study the same group investigated whether MSCs of MRL mice have better regenerative capacity than the MSCs of C57Bl6 mice25. Bone marrow derived MSCs from MRL/MpJ mice appeared to form more colonies but did not have a better differentiation capacity, which might indicate better regenerative capacity. Moreover, MSCs from both mouse strains injected after intra-articular fracture in the knee of the badly healing C57Bl6 mice, appeared to prevent the development of posttraumatic arthritis25. Presumably, the injected MSCs caused a mild increase in anti-inflammatory cytokine levels in the synovial fluid. This anti-inflammatory effect of MSCs on joint tissues has been shown in vitro with human bone marrow derived MSCs26 as well as human adipose derived MSCs27, making stem cells still a promising cell source for the treatment of osteoarthritis. The number of clinical studies using stem cells beyond the stage of case series is increasing and dose response safety studies have been conducted28. However, inflammation is not only preventing tissue regeneration. Attraction of more macrophages by the recruitment agent SEW2871 and PRP hydrogel, lead to more bone regeneration in rats29. A crucial role for macrophages in tissue regeneration was also demonstrated in salamanders30. Early after amputation of the salamander limb, anti-inflammatory macrophages appear along with the pro-inflammatory macrophages. Removal of macrophages using clodronate liposomes prevented limb regeneration. More knowledge about the dual role of inflammation in tissue regeneration is required. Targeting the timing of macrophage presence and macrophage phenotype in mammalian wound healing processes will probably lead to enhanced outcome of tissue repair opening new possibilities in the regeneration of tissues in inflamed environment such as an osteoarthritic joint.
2015
Concluding remarks Fast progression in the fields of stem cell biology, growth factors and biomaterials and the improving multidisciplinary relations between biologists, pathologists, immunologists, engineers and clinical scientists may in the future offer possibilities to regenerate joint tissues when prevention of osteoarthritis has failed.
Author contribution Gerjo van Osch searched the literature, selected the articles, summarized the results and wrote the manuscript.
Conflict of interest The author has no conflicts of interest.
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