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Keywords:

  • cell biology;
  • matrix metalloproteinase;
  • molecular biology;
  • orthopaedics

Abstract.

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Cartilage repair is a very successful pioneering area of regenerative medicine in which techniques of in situ regeneration and cell and tissue transplantation dominate over cell-free approaches to generate durable neocartilage. This review concentrates on advantages and limitations of mesenchymal stem cell (MSC)-based cartilage repair strategies induced by marrow stimulation. Detailed knowledge on the biology of MSC will be discussed in light of the requirements for MSC recruitment, retention, proliferation and chondrogenic differentiation. An improved microenvironment with timely correlated signals from biomaterials, growth factors, proteases, adjacent cartilage and subchondral bone may be key to a third generation of techniques to regenerate hyaline cartilage.


Cartilage repair: a pioneering area of regenerative medicine

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Mature joint cartilage is free of blood vessels and enervation, composed of a proteoglycan and collagen type II-rich extracellular matrix, and only about 5% of the tissue volume is occupied by cells. These chondrocytes are spherical, embedded in lacunae filled with pericellular matrix, and have no contact to the distant neighbour cells. Although human cartilage can reach a thickness of up to 7−8 mm, its supply with nutrients and oxygen is constrained to diffusion which is, however, facilitated by compressive cyclic loading providing a pumping mechanism during joint movements. Thus, chondrocytes are isolated cells, adapted to a low basic metabolic rate, and obtain functional information only through mechanical loading and diffusible humoral factors. In case of cartilage injury, this could mean that they may not sense a problem, are unable to leave their territory through the dense matrix to fill the defect, and have little potential to increase their metabolic rate to regenerate the tissue. This makes articular cartilage a tissue with an almost absent intrinsic repair capacity in most in vivo situations, although tissue culture demonstrates that in vitro conditions may reactivate a significant regenerative potential for juvenile tissue and articular chondrocytes.

Focal cartilage defects have been noted in up to 63% of patients undergoing arthroscopy of the knee [1, 2] and fortunately, the majority of them remain symptomless for quite a long time. Symptomatic lesions, however, can result in significant pain and morbidity, and a prospective clinical study demonstrated that the risk of patients with a cartilage lesion to progress to osteoarthritis is enhanced more than fivefold [3]. Such patients, thus, require a treatment filling the gap between palliation and resurfacing via arthroplasty. This need and the detailed knowledge we have about the functional elements of cartilage tissue, made cartilage repair a pioneering and very successful area of regenerative medicine.

In situ regeneration and cell therapy: two basic biological repair strategies

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

First approaches to heal cartilage by in situ regeneration date back as early as 1959 [4]. The Pridie technique was directed at recruitment of bone marrow cells to cartilage defects by drilling small holes into the subchondral bone marrow space which underlies regions of damaged cartilage. It was refined later on by reducing the size of the perforations to be called microfracture technique which is now a frequently performed and well studied procedure [5].

The first cell therapy named autologous chondrocyte transplantation (ACT) was initiated in 1994 and relied on combined implantation of an autologous periosteal flap and expanded articular chondrocytes [6]. Expanded autologous chondrocytes injected in suspension underneath the defect-covering periosteal flap is supposed to regenerate the destroyed cartilage by rapid production of a cartilaginous extracellular matrix. Limitations are the creation of new cartilage defects to harvest the chondrocytes and the requirement for cell expansion to obtain sufficient cell numbers to fill large defects. Dedifferentiation of the chondrocytes to a fibroblast-like phenotype during expansion eventually destroys their ectopic cartilage formation capacity [7] and may contribute to the fact that newly synthesized cartilage often consists of fibrous instead of hyaline tissue. Whether transplanted cells contribute physically to the new repair tissue is still a matter of debate. The technique is now in its fourth generation of development [8] and advantages, limitations, and improvements of ACT have been addressed by several recent reviews [9–12]. Whilst in the US, more than 25 000 patients were treated by marrow-stimulating techniques and about 1500 by ACTs in 2007, ACT is applied more frequently in Europe. Manifestation of osteoarthritis is usually an exclusion criterion for both techniques.

These two cell-based biological repair strategies are complemented by cell-free approaches like the filling of cartilage defects by synthetic or natural scaffolds which are classified as medical devices, but little data are available about clinical success (see McNickle et al. for review [13]). Alternatively, small to medium-sized defects can successfully be treated by mosaic-type transplantation of autologous osteochondral cylinders which are harvested from peripheral areas of the same joint [14]. Disadvantages of this technique include the significant donor-site morbidy by introduction of new nonhealing cartilage defects and the limited availability of such tissue.

This review will concentrate on advantages and limitations of mesenchymal stem cell-based cartilage repair strategies induced by marrow stimulation and discuss new knowledge on the biology of mesenchymal stem cells in light of the requirements for cartilage in situ regeneration to allow further improvements of today’s efforts to regenerate hyaline cartilage.

Bone marrow-stimulation by subchondral bone perforation

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Diagnostic arthroscopy is routinely indicated on suspicion of an articular cartilage defect. If a lesion below 1–4 cm2 is confirmed, the first choice of treatment is often microfracturing, the penetration of the subchondral bone plate by creation of small holes. Bleeding from the subchondral bone spaces yields a blood clot which is thought to develop into a favourable microenvironment called superclot capable of stimulating attraction, proliferation and chondrogenic differentiation of mesenchymal stem cells (MSC) coming from the bone marrow.

The advantage of a marrow-stimulating technique is that several of the main goals of cartilage repair are fulfilled: it represents an easy, simple, minimally invasive, low-morbidity, single-stage procedure, and is a cost-effective technology with few associated complications and with a high capacity for creation of durable cartilage repair tissue [15]. However, clinical results are age-dependent. In young athletes and young patients, marrow stimulation resulted in improvements in up to 75% of patients after 5 years of follow-up [16, 17]. The repair tissue seen was fibrocartilage or a hybrid of hyaline and fibrous cartilage. This was used as an explanation for the observation of another study that good short term results may be followed by deterioration starting around 18 months after surgery [18]. However, a randomized clinical trial comparing ACT with microfracture did not find a correlation between histological quality of repair tissue and clinical outcome after 5 years of follow-up [19]. Microfracture provided satisfactory results in 77% of the patients at 5 years, just like ACT, and there was no significant difference in clinical and radiographic results between the two treatment groups. There was, however, a tendency for patients with smaller defects in the microfracture group to have better clinical results. This tendency was not present in the ACT group.

Limitations of marrow-stimulating techniques

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Clinical observations and theoretical considerations pointed towards several possible limitations of marrow-stimulating techniques. The nonadhesive properties of a cartilage surface and the softness and shrinking of the superclot may lead to only partial defect filling and facilitate early loss of repair tissue from the cartilage lesion. To avoid this, the treatment was recently advanced into a matrix-supported technique in which the perforated defect was additionally stabilized with a biomaterial. Covering the microfractured lesion with a collagen typeI/III scaffold was called autologous matrix induced chondrogenesis (AMIC) [15, 20]. It was developed to allow treatment of larger defects by microfracturing and is used as alternative treatment to ACT when cell-engaged procedures are not indicated or cannot be used for financial reasons. The results of first clinical studies on such biomaterial-supported 2nd generation microfracturing techniques are awaited.

Biomaterial-based clot stabilization by polyglycolic acid scaffolds was a prerequisite to achieve cartilage repair in a preclinical cartilage repair study in sheep. As no repair tissue formed in the classical microfracture group in this study, the general value of this animal model for the human situation should be questioned [21]. In another sheep study a hydrogel was applied with the aim to stabilize the fibrin clot. In this ‘BST CarGel’-called technique, chitosan-glycerol phosphate based scaffolds resulted in more hyaline-like fill compared with microfracture alone [22] and phase 3 clinical trials are on their way to estimate the potential of this technique for clinical application.

Beyond clot fragility, a second possible limitation of marrow-stimulating techniques is the expected low incidence of MSC in bone marrow. Only about 7–10 MSC per 1 × 106 mononucleated cells can be isolated from bone marrow aspirates [23–25]. Thus, <100 MSC would be contained in an initial clot of several millilitres filling a large defect. For comparison, the cartilage tissue in a 5 cm2 defect of 4 mm thickness would contain about 10–20 million chondrocytes. To enhance the initial number of repair cells and induce their efficient in situ proliferation could be a means to speed regeneration and improve final results. A third very complex question is, whether the microenvironment in the superclot is adequate to allow and optimally support chondrogenesis of MSC. Factor composition in the forming blood clot is ill-defined and may not be ideal to support attraction, proliferation and chondrogenesis of new cells as angiogenic growth factors and fibrogenic factors are predominant. This may favour a fibrous or even bony regenerate which is consistent with observations from clinicians [26] and from animal models [27] that fibrocartilage forms and that the subchondral plate may advance into the defect during osteochondral repair [28]. It is likely that variable healing results, fibrous nature of repair tissue, reduced biomechanical resistance and limited durability of the regenerate are related to suboptimal attraction, retention, proliferation and differentiation of mesenchymal stem cells.

Detailed knowledge on the biology of MSC is now available to design 3rd generation techniques based on the step-wise requirements for cell attraction, proliferation and chondrogenic differentiation. This will be discussed in the next paragraphs with the aim to open new avenues to further improve MSC-based in situ regeneration strategies for cartilage defects.

Mesenchymal stem cells and cartilage in situ regeneration

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Definition of MSC

Mesenchymal stem cells are stem cells derived from somatic tissue which can be differentiated into mesenchymal lineages such as bone, cartilage and fat. Multipotent cells with chondrogenic differentiation potential have been identified not only in bone marrow, but also in other joint-related tissues such as synovial membrane [29, 30], infrapatellar fat pad [31] and periosteal tissue [32]. Accumulating evidence suggests that MSC are found in a subendothelial location in bone marrow stroma [33] and in a perivascular location in adipose tissue [34]. The presence of a set of cell surface markers (CD13, CD29, CD44, CD54, CD73, CD 90, CD105 and CD166) in the absence of hematopoietic markers (CD34 and CD45) characterizes these cells. However, this marker profile is not sufficient to define multipotency, as determined mesenchymal cell types like chondrocytes or fibroblasts may stain for the same markers. For functional characterization, testing of the in vitro differentiation capacity of MSC into the osteogenic, chondrogenic and adipogenic phenotype is, thus, indispensable.

Requirements for MSC-based in situ cartilage repair

To initiate any regeneration based on MSC activity, the cells first have to be recruited to the site of damage (Fig. 1). Second step is adhesion to a local matrix followed by activation and extensive proliferation to provide the necessary high numbers of chondroprogenitor cells to build up new tissue. In step 3, the cells need to switch from expansion to chondrogenic matrix production by induction of chondrogenesis to build up the shock absorbance and gliding characteristics for proper tissue function. The seamless integration with neighbouring cartilage and bone tissue depends on successful crosstalk between new and old tissue, and an instructional capability to guide neighbouring cells. For durable cartilage repair, the tissue eventually needs to regenerate a tidemark, adapt to biomechanical loading and build up a balanced tissue homeostasis. Actual knowledge on these steps is here summarized together with open questions to stimulate advancements for an upcoming 3rd generation of in situ cartilage repair techniques.

image

Figure 1.  Step-wise requirements for cartilage in situ-regeneration.

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Attraction and activation of MSC

Cell migration is a prerequisite for development from conception to adulthood and plays a major role in regeneration of all tissues. Even articular chondrocytes, which are encased in a dense matrix throughout their life, can show cell motility when transferred in vitro. About 1/3 of freshly cultured chondrocytes are motile, move at a speed of 5−50 μm h−1 and, thus, are slow compared with other cells (20-720 μm h−1) [35]. A number of studies demonstrated that chondrocytes migrate under the action of different stimuli on or within planar and 3D matrices. Attracting factors include bone morphogenetic factors (BMPs) [36], hepatocyte growth factor (HGF) [37], insulin-like growth factor (IGF-1) [38], transforming growth factor (TGF-β) [38], platelet-derived growth factor (PDGF) [39], fibroblast growth factor (FGF) [40, 41], fibronectin [38], fibrin [42] and collagen type I. Results, however, remain contradictory for some of these factors as testing of human chondrocytes revealed no effects for BMP-2, BMP-4, BMP-7, IGF and TGF-β in other studies [43].

But, what are the best factors to attract more MSC to a cartilage lesion? Recent work has established that MSC are not largely distinct from chondrocytes regarding the panel of factors capable to attract the cells in vitro (Table 1). Directed in vitro migration was stimulated by many growth factors including PDGF, EGF, TGF-β, IGF-1, HGF, BMP-2, BMP-4, [44–46] and VEGF [47], with most potent effects observed for IGF-1, PDGF [44, 45, 48] and VEGF [43]. Several chemokines had rather limited effects. Interestingly, combination with FGF-2 or thrombin suppressed the PDGF-BB-promoted migratory activity of MSC [44]. All tested factors were further effective in stimulating the migration of fibroblasts, except for thrombin alone, which selectively enhanced the migration of MSC but not fibroblasts [44]. Altogether, this suggests that fibrin, PDGF and other factors contained in a natural blood clot are highly chemoattractive for MSC, although the effect of defined combinations has not been evaluated so far. Augmentation of a clot-stabilizing matrix with a potent chemoattractive factor like PDGF or EGF may be an attractive way to further enhance cell numbers early after microfracturing. A quick release kinetics within hours and days from the biomaterial is desired for this first step of healing. Unfortunately, no 3D in vitro model in a biomatrix has yet been applied to test chemoattraction of human MSC under more natural conditions and no adequate animal model has been used to dissect the factor requirement of progenitor cell recruitment from bone marrow into cartilage defects. Most likely, the presence of synovial fluid and the joint loading initiated pumping mechanism will strongly affect attraction and retention of MSC. A search for superior factor combinations to enhance and speed up MSC attraction, and the best retention matrix to keep cells local, thus, is an important topic for upcoming studies.

Table 1.   Factors stimulating mesenchymal stem cell migration and proliferation
FactorStimulation of MSC
MigrationProliferation
  1. This list is not exhaustive concerning cited references.

PDGFMishima 2008 [43] Ozaki et al. 2007 [44] Ponte et al. 2007 [45] Fiedler et al. 2004 [48]Kuznetsov et al. 1997 [54] Cassiede et al. 1996 [105] Gronthos and Simmons 1995 [106]
EGFOzaki et al. 2007 [44] Fiedler et al. 2002 [46]Kuznetsov et al. 1997 [54] Gronthos and Simmons 1995 [106]
IGFMishima 2008 [43] Ozaki et al. 2007 [44] Ponte et al. 2007 [45] Fiedler et al. 2006 [107] 
VEGFFiedler et al. 2005 [47] Mishima 2008 [43] 
HB-EGFOzaki et al. 2007 [44]Krampera et al. 2005 [108]
TGF-αOzaki et al. 2007 [44] 
TGF-βMishima 2008 [43] Fiedler et al. 2002 [46]Jian et al. 2006 Kuznetsov et al. 1997 [54] Cassiede et al. 1996 [105]
FGF-2Ozaki et al. 2007 [44]Bianchi et al. 2003 [109] Tsutsumi et al. 2001 [110] Martin et al. 1997 [111] Kuznetsov et al. 1997 [54]
FGF-4 Farréet al. 2007 [112]
HGFOzaki et al. 2007 [44] Fiedler et al. 2002 [46] 
BMP-2Mishima 2008 [43] Fiedler et al. 2002 [46] 
BMP-4Mishima 2008 [43] Fiedler et al. 2002 [46] 
Dkk-1 Gregory et al. 2003 [113]
IL-17a Huang et al. 2006 [114]
HMBG1Meng et al. 2008 [115] 

Proliferation of MSC

Proliferation of MSC is the second important step to rapidly enhance the cell numbers in the repair tissue. As the replicative lifespan of MSC is, however, not unlimited and telomerase activity is absent or low [49–51] this can only ground on the sufficient attraction of initial MSC numbers to the defect. During embryonal development proliferative chondroprogenitors are densely packed and condensed to an area in which cartilage tissue is forming. The high cell numbers may be needed to deposit the vast amount of extracellular matrix characterizing this tissue. Low cellularity of articular cartilage is rather a late phenomenon during tissue formation and may have developed in adaptation to its biomechanical competence and the extremely slow turnover of its extracellular matrix. Proteoglycan turnover in cartilage is up to 25 years and collagen half-life was estimated to range from several decades up to 400 years [52]. Based on the assumption that embryonal traits should best be recapitulated during tissue regeneration, early defect filling tissue should contain densely packed proliferating chondroprogenitors. This would mean that rather hundreds of millions of new cells are needed per cm3 during this step in the defect to allow for optimal chondrogenesis and rapid extracellular matrix production. At a later phase, cell numbers could decline to a density of 5–10 mio cells per gram tissue, when homeostasis is the left over task for the cells in the fully regenerated tissue.

This points towards an outstanding need for highly efficient transient induction of proliferation, a typical feature ascribed to adult stem cells. Growth requirements of human MSC are distinct from those of other species [53] and many factors have been identified as potent mitogens (Table 1). PDGF-BB, EGF and TGF-β have been regarded as the most important amongst them [54]. As they induced the migration of MSC and have at the same time the potency to enhance their proliferation, the migration and proliferation steps of MSC can take place simultaneously in vivo. However, whilst nutrients and oxygen are almost unlimited in tissue culture, a rapid supply of cells with O2 and nutrients may be more restricted in a cartilage defect and depend on the distance to and conditions found within the subchondral bone. Histology of early human cartilage repair tissue demonstrates that indeed cartilage differentiation initiates in contact with subchondral bone [55] whilst upper regions remain fibrous for quite a long time and may need to mature over years [56]. Furthermore, earliest chondrogenesis is often seen in areas where active remodelling of the subchondral bone plate occurs and, thus, enhanced nutrition and a higher anabolic rate of the cells can take place. Beside strong mitogenic factors used for augmentation of a clot-stabilizing biomaterial, the access to optimal nutrition in the course of tissue remodelling may indeed be a limiting aspect of cartilage regeneration techniques. Enhanced remodelling of microfractured compared with unopened subchondral bone areas is likely, but quantitative and localization-dependent studies have so far not been reported.

Chondrogenesis of MSC

A switch from proliferation to differentiation of MSC (Fig. 1) is an inherent tendency of MSC which is influenced by cell density in culture. A spontaneous osteofibroid differentiation of bone marrow derived MSC is evident in over dense tissue culture flasks where a mineralising matrix is deposited in spite of supply with growth (and not differentiation) medium. Phenotypic chondrogenic in vitro differentiation is much more demanding than successful osteogenesis or adipogenesis. It is usually not successful in monolayer culture, involves serum withdrawal and supplementation with ascorbate, and requires dexamethasone and TGF-β as indispensable factors for human cells [57]. The microenvironment seems crucial to reproducibly induce successful chondrogenesis and beside hormones, vitamins and growth factors, the embedding in a biomatrix, the presence and activity of proteases and biomechanical loading are of influence. All these aspects may be important on the way of optimizing in situ cartilage repair strategies.

Growth factors for chondrogenesis

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Any of the TGF-β subtypes (TGF-β1, TGF-β2, TGF-β3) is an equally active chondrogenic factor and rather lot- than subtype-specific differences seem to exist. Whilst members of the BMP family like BMP-2, BMP-4, BMP-6 and BMP-7 act synergistically to TGF-β and enhance matrix deposition, they are themselves not sufficient to drive in vitro chondrogenesis of human MSC at low (10 ng mL−1) [58] and high (400 ng mL−1) concentrations [59] in classical pellet culture with dexamethasone supplemented medium. The same applies for IGF-1 and FGF-1 whilst FGF-2 and PTHrP suppress TGF-β-driven chondrogenesis [58]. Bone marrow-derived MSC from most donors show successful differentiation in chondrogenic medium containing 10−7 mol L−1 dexamethasone and 10 ng mL−1 TGF-ß. MSC derived from adipose tissue or synovial tissue usually require the addition of BMP-6 beyond. This factor seems to be the most potent of the BMP family for support of chondrogenesis [58, 60, 61]. Augmentation of a clot-stimulating matrix with strong chondrogenic factors seems to be an attractive means to stimulate chondrogenic matrix deposition during cartilage in situ regeneration. A delayed release kinetic starting a few days after implantation and lasting over the first weeks is desired. A natural binding capacity of TGF-β to fibrin glue has been established and its release was slow enough to last over more than 10 days in vitro [62]. In vitro augmentation of fibrin gel with TGF-β was sufficient to drive chondrogenesis of human MSC as successfully as 6 weeks of TGF-β supplementation by culture medium (A. Dickhut, K. Martin, R. Lauinger, C. Heisel and W. Richter, unpublished data).

In response to chondrogenic conditions, a broad shift in gene expression is noted [63, 64] and different phases of induction have been defined [65, 66] during which typical cartilage marker molecules like collagen 2, aggrecan, COMP, decorin and biglycan are induced. Importantly, however, the detection of mRNA for these molecules is no sufficient indication for successful phenotypic differentiation. Only deposition of collagen type II protein and proteoglycans in the extracellular matrix correlates well with the phenotypic change of cells from a fibroblastoid to a chondroid phenotype [58] making histochemical protein and proteoglycan detection one of the most important methods for evaluation of chondrogenesis in vitro and in vivo.

Biomaterials: a facilitating environment for MSC chondrogenesis

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

The value of biomaterials is their capacity for rapid defect filling, enhanced persistence to effect repair, the chance for even distribution of cells in an enhanced volume and provision of an active environment which could allow for local release of molecules stimulating repair.

As aggregation of chondroprogenitors and the formation of cell–cell and cell–matrix contacts is an important step during embryonal chondrogenesis [67], the value of biomaterials keeping the cells apart from each other was in question. Chick limb cells that aggregate first will be the first to differentiate and a reduced rate of cell clustering and cell to cell contact paralleled a reduction of cell recruitment into the cell differentiation program [68]. Is a fibrin clot containing equally dispersed cells at low to medium numbers then a rather suboptimal microenvironment for de novo chondrogenesis of MSC? Surprisingly, the cell–cell contacts possible in high density pellet culture seem to be no prerequisite for chondrogenesis of MSC. Gel-like biomaterials made of collagen or fibrin, in which MSC are kept apart from each other by the biomaterial, rather promoted the chondrogenic differentiation capacity of MSC in vitro compared with pellet culture [69]. Beside the enhanced deposition of collagen and proteoglycans in the constructs, the biomaterials were even capable to rescue the differentiation potential of MSC from donors which showed no successful chondrogenesis in pellet culture or from cells which were exposed to suboptimal growth factor conditions.

Collagen and fibrin-based gels are, however, subject to strong shrinking during chondrogenesis [69] which points towards an increasing risk of partial defect filling and loss of a superclot after microfracturing during progress of chondrogenic differentiation. To be able to avoid this, a clinically applied solid collagen type I/III matrix, used for example in the AMIC technique, was also tested for a support of chondrogenesis of MSC. Fibrin sealant was applied for homogeneous cell seeding in the matrix and was intended to simulate the natural fibrin clot. Furthermore, fibrin glue is frequently used for fixation of biomaterials in cartilage defects [70, 71] and for these reasons its effect on chondrogenesis of MSC may be important. The form stability of the collagen I/III matrix prevented strong shrinking during 6 weeks of in vitro chondrogenesis and proteoglycan deposition was significantly enhanced beyond values for the fibrin sealant alone or matrix-free pellet cultures (A. Dickhut, K. Martin, R. Lauinger, C. Heisel and W. Richter, unpublished data). Thus, current clinically applied biomaterials have the capacity to rather facilitate than reduce chondrogenesis of MSC, indicating that 2nd generation matrix assisted microfracturing techniques may be able to enhance chondrogenic differentiation of human MSC and thus improve cartilage regeneration. However, not all biomaterials showed in vitro results beyond those obtained with MSC in biomaterial-free pellet culture [72, 73], underlining the need for careful material design and testing before use in the respective setting. Particularly attractive for future testing seems the use of biphasic implants composed of a bioresorbable scaffold combined with a gel, in which both, scaffold and gel, may be augmented with desired growth factors to improve the results.

The role of proteases in chondrogenesis

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Commercial fibrin sealants usually contain fibrinolysis inhibitors like aprotinin to prolong the stability of the adhesive and prevent premature liquefaction which is observed for self-prepared autologous fibrin glue [74]. The activity of multiple proteases is crucial for cell migration, fibrin clot/biomaterial remodelling and growth factor activation. Furthermore, expression and regulation, especially of matrix metalloproteinases (MMPs) may be important for the transition of MSC to mature chondrocytes [75, 76]. Proteases may, thus, represent attractive targets to influence any of the steps represented in Fig. 1 and their modulation could help to find optimized conditions for cartilage repair.

When a panel of MMPs (MMP-1, -2, -3, -8, -9, 10, -11, 13) was tested for expression in expanded human MSC, MMP2 mRNA and protein was most abundantly expressed. No significant regulation of MMP2 was evident during chondrogenesis according to quantitative RT-PCR analysis. In contrast, MMP10, MMP11 and MMP13 mRNA was significantly upregulated during differentiation and an induction and secretion of MMP13 into culture supernatants characterized the late phase of chondrogenesis [77]. MMP13 induction is characteristic for terminally differentiating chondrocytes in the growth plate but not for development of articular chondrocytes which suggested that cells may undergo a program related to endochondral differentiation rather that articular cartilage formation.

Whilst aspartate protease-, cysteine protease- and serine protease-inhibitors had little effect on chondrogenic in vitro differentiation of MSC, several broad-spectrum MMP inhibitors suppressed initiation and progression of chondrogenesis and, thus, early and late aspects of differentiation in a dose-dependent manner [77]. Stabilization of fibrin clots by pepstatin, leupeptin, or aprotinin is, thus, permissive for chondrogenic differentiation of MSC, whilst the inhibition of MMPs by piperazine-based or hydroxamate-based inhibitors is deleterious and, thus, worth of further detailed investigation. In conclusion, chondrogenic differentiation of MSC is a sophisticated process in which catabolic enzymes are capable to directly influence cellular fate. The clinical approach to reduce catabolism as a means to delay cartilage matrix degradation in a damaged joint [78] may therefore, in case of MMPs, be at risk to block a progenitor cell driven chondrogenic repair response to build up new cartilage. MMP inhibition may, thus, be not attractive to enhance the results of MSC-based cartilage repair strategies.

Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Common protocols of in vitro chondrogenesis produce MSC-derived chondrocytes which express collagen type I and X beside collagen type II [57]. Furthermore, activation of alkaline phosphatase (ALP) activity and upregulation of MMP13 [58, 66, 79] are reminiscent of matrix calcification and terminal differentiation like in endochondral bone formation and suggest that rather transient than stable articular chondrocytes are generated during in vitro chondrogenesis. Indeed, subcutaneous transplantation of MSC-derived chondrocytes in pellets resulted in calcification and microossicle formation in the transplants whilst expanded articular chondrocytes remained negative for COL10A1, ALP and MMP13 and produced stable ectopic cartilage under the same conditions [66].

Synovial membrane-derived MSC (SMSC) were discussed as particularly attractive cells for cartilage repair due to their close vicinity to cartilage, their high chondrogenic capacity [80] and easy availability during arthroscopy. As SMSC are derived from a joint tissue in which calcification is usually suppressed, it was speculated that they may have a lesser tendency for mineralization and may, thus, be a more attractive source for cartilage repair than bone marrow. Indeed, in a direct comparison, SMSC-derived chondrogenic pellets produced significantly lower ALP activity during chondrogenesis than bone marrow-derived or adipose tissue-derived MSC, and pellets with low ALP activity did not undergo matrix calcification after ectopic transplantation in vivo. However, this did not add to the ectopic stability of these transplants, as the pellets either lost their in vitro acquired deposition of collagen type II and dedifferentiated, or even degenerated at ectopic sites [61, 81]. Enhanced MMP levels detected in these samples may be a possible explanation for such effects [61]. This demonstrated that the transplanted cells kept a high versatility during 6 weeks of chondrogenic differentiation and responded to the shift from chondrogenic medium to in vivo exposure under nonjoint like conditions by changing their differentiation. This indicates that we not only need appropriately improved in vitro induction protocols for chondrogenesis of MSC, but also have to find mechanisms to lock the cells in a reached differentiation state.

One possibility for stabilization could be the establishment of appropriate epigenetic patterns in the DNA of the cells. Methylation of cytosine at CpG sites (sometimes called the fifth base in the genome) can lock DNA regions in an inactive state, thereby permanently blocking expression of the affected genes in this cell type [82]. Whilst promotor regions of human COL2A1 showed no apparent differences in methylation status between articular chondrocytes and MSC, such differences were identified in COL10A1 regulatory regions in correlation to collagen type X expression. Epigenetic phenomena like reduced methylation in the COL10A1 promotor and its further demethylation during chondrogenesis may, thus, contribute to facilitated induction of this hypertrophic marker molecule in MSC [83]. Until more knowledge is available on how to stabilize cell differentiation, an in vitro production of chondrocytes from MSC before transplantation is not only of high effort, but seems also unattractive. Next steps are to investigate whether an orthotopic microenvironment in a cartilage defect may be able to contribute to such a stabilization and eventually lock the cells in the adequate state of cartilage matrix production. Sensitive labelling and re-identification techniques for such cells are an important prerequisite for such studies.

Integration of repair tissue

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Wounding of cartilage usually induces a zone of cell death at the sites of initial mechanical trauma which may penetrate into the tissue over time [84]. This zone of cell death likely hinders integration between neocartilage and existing tissue due to sub-optimal matrix production and lack of cells. Whilst vertical integration with the subchondral bone plate and reconstruction of the tidemark is routinely achieved through distinct healing methods [85], lateral integration is a chronic problem in cartilage repair. Though initially repair tissue may be fused to wound edges, lateral integration was lost over time in long term animal models [27] and the biomechanically compromised tissue is therefore prone to mechanical failure.

Several groups reported about inhibitory phenomena at the interface between growing neocartilage and native intact cartilage in experimental in vitro models [86, 87] but the nature of the refractory signal is open to question. One key molecule may be PRG4/superficial zone protein/lubricin produced in synovial membrane and the superficial zone of cartilage [88, 89]. This glycoprotein provides important lubrication of congruent articular surfaces under conditions of high contact pressure. Using a disc/ring composite model of cartilage integration, the adhesive strength of control composites was 10-fold higher than that of composites continuously cultured in the presence of lubricin [88]. Components of the synovial fluid and the articular surface, thus, may have an inhibitory role in neocartilage integration.

Strength of integration may further depend on the age and metabolic activity of the tissue. When integration was studied between foetal, neonatal and adult cartilage, an inverse correlation with age was obvious, which was a reflection of differences in the biosynthetic capacities of the compared samples [90, 91]. Thus, the use of more immature cells had obvious benefits for integration, which argues in favour of MSC-based as opposed to chondrocyte-based repair strategies.

Amongst several options developed to enhance the integration of new tissue into cartilage defects, enzymatic removal of glycosamionogylcan chains as an intrinsic barrier for cell migration or of collagen by collagenase have been promoted. These strategies ground on the expectation of quick reconstitution of the damaged surfaces by the viable cells and are detailed in a recent review [92]. In general, they seem more adequate to enhance integration of mature tissue engineered grafts and may be less relevant for in situ regeneration.

Interaction of MSC with cartilage and subchondral bone

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Beyond inhibitory paracrine interactions between cartilage surface and repair cells, like those suggested for lubricin and similar molecules, subchondral bone and adjacent cartilage may otherwise influence incoming cells regarding cell metabolism and commitment. Chondrocytes from chicken have been shown to provide osteogenic signals to a mouse mesenchymal stem cell line in indirect coculture [93] and human intervertebral disc-derived cells stimulated MSC viability [94] and expression of chondrogenic marker genes [95, 96], however, only in direct coculture allowing cell–cell contacts. Studies on chondrocytes and osteoblasts released from the tissue, however, seem less informative for several reasons. First, direct cell–cell contacts will rarely occur between repair cells and tissue cells as chondrocytes are immobilized in a dense matrix and thus communication is mainly confined to paracrine signals. Secondly, release from the extracellular matrix or outgrowth from bone chips already shifts gene expression of cells towards dedifferentiation [97] and the natural balance between anabolic and catabolic factors stored in the matrix is lost. Another limitation of several coculture studies is that cells of distinct species were combined [93, 98] and that MSC from rat [55, 99], calf or goat [98] were studied, which have a higher intrinsic tendency to undergo spontaneous osteochondral differentiation. A shift of these MSC to 3D conditions is usually sufficient to drive chondrogenesis in the absence of inductive growth factors [55, 99, 100], which is very different to human cells. Thus, an anabolic rather than chondroinductive stimulus may suffice to explain the described effects in those studies. Since data on immortalized human chondrocytes and immortalized human MSC may also be regarded in the light of such effects [101], well designed in vitro studies on the paracrine effects of cartilage or bone tissue on human MSC are awaited to dissect inhibitory and stimulating influences.

Integration of in vivo signals: superior chondrogenesis at orthotopic sites

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

The interplay between attracted repair cells or transplanted MSC with subchondral bone was also studied in a cartilage chondral defect model in minipigs [55]. Extensive remodelling of the subchondral bone plate correlated with signs of vigorous induction of chondrogenic differentiation of repair cells in the defect (Fig. 2A). Interestingly, lesions in which this remodelling was not initiated for whatever reason, contained mostly fibrous nonchondrogenic tissue at 8 weeks after surgery (Fig. 2B). It is conceivable that penetration of subchondral bone by microfracturing will more likely induce such a tissue remodelling response compared with careful preparation of a chondral lesion without induction of any bleeding, which is recommended for ACT [102]. Studies are awaited in which this interesting question will be carefully evaluated.

image

Figure 2.  Histology of repair tissue after transplantation of autologous MSC into a chondral defect in the knee of minipigs. A bottom layer of calcified cartilage was not removed at defect preparation in all defects of the study. (a) At 8 weeks after surgery a vigorous remodelling of the subchondral bone plate had occurred in some defects which correlated with induction of chondrogenesis in repair cells in bone-close areas. Immune histology for collagen type II demonstrated that differentiation was most advanced in contact to bone and lateral cartilage tissue. (b) Little chondrogenic differentiation occurred in defects, in which the remodelling of the subchondral bone plate was less advanced for unknown reasons, and in which the bottom of the former defects still contained the calcified cartilage layer.

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Remarkably, the orthotopic differentiation of progenitor cells in the cartilage defect in this study resulted in appropriate induction of collagen type II-positive chondrocytes whilst collagen type X-positive areas were confined to the contact zone with subchondral bone [55]. Compared with in vitro chondrogenesis, significantly lower COL10A1/COL2A1 and MMP13/COL2A1 ratios were obtained in vivo, and a spatially organized repair tissue with upper fibrous, intermediate chondrogenic and low layer hypertrophic appearance was formed. This indicated that in vivo signalling molecules and biomechanical stimuli provided a much more appropriate environment for progenitor cells to differentiate than in vitro chondrogenesis. It furthermore suggested that inhibitory signals may come from the opposed cartilage surface and synovial fluid to dominate the surface area of fibrous repair tissue, whilst chondroinductive signals were provided from a remodelling and thus anabolically active subchondral bone area. The lowest cartilage layer is responsible for load transmission from cartilage into bone. Application of biomechanical loading during chondrogenesis of MSC stimulated cartilaginous matrix production in tissue engineering applications [103, 104] underlining the importance of mechanical signals for tissue guidance during repair. Obviously, current in vitro protocols of MSC chondrogenesis seem to recapitulate only conditions of the deepest cartilage repair zone in contact to bone as collagen type X is always detected. Spatially oriented or sequential protocols will now have to be elaborated with the hope to identify factors or conditions active in the middle region that could then be used to enhance and speed up chondrocyte differentiation also the in upper areas.

Conclusions

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References

Because of the high costs associated with MSC isolation and expansion, the lack of proper in vitro protocols for induction of chondrogenesis in the absence of hypertrophy in MSC, and of adequate means to stabilize or lock a desired differentiation stage before implantation, the in situ recruitment of MSC to a cartilage defect site seems currently more attractive than their implantation.

The advantage of microfracturing is that it represents an easy, simple, minimally invasive, low-morbidity, single-stage procedure which is cost-effective and associated with few complications and a high capacity for creation of durable repair tissue. Younger and active patients (<40 years) with isolated traumatic lesions ≤4 cm2 on the femoral condyles have the best long-term results when the classical technique is applied. To expand application to larger defects and to achieve better results in older individuals and in the long-term, a second generation of biomaterial-supported marrow-stimulation techniques was recently introduced to circumvent fragility and loss of the therapeutic superclot from the defect. New knowledge in mesenchymal stem cell biology can now be applied to adapt the technique to the step-wise requirements of cell recruitment, adhesion and retention, proliferation, and chondrogenic differentiation of MSC, to generate a shock absorbing matrix with high gliding characteristics which is of enhanced durability. New augmentation techniques for defect-filling biomaterials could combine fast release kinetics of strong chemoattractive and mitogenic factors, like PDGF and EGF, with delayed release of strong chondrogenic factors like TGF-ß and BMP-6, to optimize matrix deposition. As limited nutrition, suppression of matrix catabolism for example by natural MMP inhibitors, and negative paracrine interactions with the opposing cartilage surface seem problematic for chondrogenesis of MSC, a strong focus should be set on stimulation of anabolism and tissue remodelling at the subchondral bone plate to oppose such influences. High remodelling activity may advance the results by promoting cell nutrition, speeding migration, proliferation and chondrogenic differentiation, thereby broadening the area of proteoglycan-rich, collagen type II positive neocartilage at the expense of the upper merely fibrous tissue layer.

Design and elaboration of such techniques based on new matrix-assisted systems will lead to a promising 3rd generation of one step cartilage intervention strategies which rely on enhanced in vivo maturation of newly attracted cells instead of extended in vitro manipulation.

References

  1. Top of page
  2. Abstract.
  3. Cartilage repair: a pioneering area of regenerative medicine
  4. In situ regeneration and cell therapy: two basic biological repair strategies
  5. Bone marrow-stimulation by subchondral bone perforation
  6. Limitations of marrow-stimulating techniques
  7. Mesenchymal stem cells and cartilage in situ regeneration
  8. Growth factors for chondrogenesis
  9. Biomaterials: a facilitating environment for MSC chondrogenesis
  10. The role of proteases in chondrogenesis
  11. Stability of chondrogenic differentiation: lessons from synovial membrane-derived MSC
  12. Integration of repair tissue
  13. Interaction of MSC with cartilage and subchondral bone
  14. Integration of in vivo signals: superior chondrogenesis at orthotopic sites
  15. Conclusions
  16. Conflict of interest statement
  17. Acknowledgement
  18. References