Clonogenic cells identified in the CFU-F assay are thought to contain a mixed population of highly proliferative MSCs and less proliferative transit-amplifying progenitors [ 14 ]. True MSCs are normally identified at the single-cell-level using limiting dilution techniques and mesenchymal lineage differentiation assays on clones expanded to a minimum of 10 PDs [ 5 , 7 , 24 ]. Single-cell-derived clonogenic cultures could not be generated from OA cartilage digests but matched OA Hoffa's fat pad digests and yielded several bi- and tri-potential clones Fig.
This suggested that although OA cartilage retained good proliferative capacity, it was likely driven by transit-amplifying progenitors and not by true highly proliferative MSCs. More recently, the expression of CD44 and CD was proposed to be characteristic for human chondrocytes with enhanced chondrogenic potential [ 31 ], but tested on our set of samples, no differences in their expression were found Table 1.
As documented previously, the number of cells expressing CD were higher in BM MSCs compared with both cartilage and fat pad-derived cultures [ 32 , 33 ].
Cartilage and Osteoarthritis
We had previously demonstrated that during culture expansion of BM MSCs LNGFR expression is lost [ 17 ]; remarkably, however, it remained on a subpopulation of fat pad-derived cultures. Histograms for a representative sample are shown. Markers—grey-shaded histograms, isotype control antibodies—empty histograms. No surface marker was found sufficiently differentially expressed to be selected as a potential indicator of observed functional differences between all the cultures studied.
Many hurdles have to be overcome with respect to therapy development for chondral defects in advanced OA. This work explored one of these issues, namely optimal sources for autologous cells for cartilage repair. The existing evidence suggests an age-related loss of chondrogenic capacity of ADDCs, the currently preferred cells for cartilage repair [ 13 , 22 ].
The data presented in this study demonstrate functional and immunophenotypic evidence for an MSC population in OA joint Hoffa's fat pad, but not in OA cartilage. Specifically, we found considerable MSC activity at the single-cell level from fat pad-derived cells but none of the cartilaginous structures. Therefore, Hoffa's fat pad could represent a useful source of regenerative cells for cartilage repair in OA. As there is evidence for some spontaneous joint repair in OA, we initially evaluated three distinct knee articular cartilage sites including chondro-osteophytes for MSC activity.
A novel aspect of this work resides in the fact that sites of neochondrogenesis N in vivo did not have a higher chondrogenic capacity compared with MN cartilage. In addition to performing clonogenic assays for resident MSCs, we deliberately assessed early passage polyclonal OA cartilage- and Hoffa's fat pad-derived cultures.
From the perspective of OA therapy development in older subjects shorter cultivation would be desirable for a number of reasons, including minimizing the risks of genetic instability. Additionally, it would reduce the time from cartilage biopsy to cell implantation, and also lessen the recognized loss of potency following expansion and implantation [ 35 ]. In these experiments, the differences in chondrogenesis between the four different OA tissues fat pad and three cartilage areas failed to reach statistical significance due to very large donor-to-donor variability in all the tissues studied, but overall, the fat pad-derived cultures had comparable and in many cases superior chondrogenesis to the cartilage-derived cultures.
It has been previously shown possible to generate single-cell-derived multipotential clones from normal young individuals in some studies [ 5—7 ] but not in the others [ 35 ], suggesting limited expandability of cartilage-derived MSCs even in health. In OA cartilage, the presence of cells with putative MSC phenotype was previously documented [ 26 , 27 , 36 ] and their frequency was suggested to be increased compared with normal cartilage, however, this remains to be proven using clonal cultures derived from single sorted cells.
The data presented in this work are in agreement with previous studies that showed some preservation of chondrogenesis in low-passage polyclonal ADDCs [ 22 , 35 ]. A potential concern of the clinical use of OA cartilage-derived cultures, however, is that the lack of true clonogenic MSCs in such grafts, which could be associated with poor long-term graft survival following in vivo implantation.
In contrast to cartilage, the ability to generate clonogenic MSCs as well as polyclonal cultures from fat pad with good chondrogenesis, even at high PDs, suggests that the fat pad has resident MSCs even in advanced OA. We have also demonstrated that extended cultivation of Hoffa's fat pad-derived cells leads to a slower loss in their chondrogenesis compared with OA cartilage. Although there are other potential sources of chondroprogenitors including bone marrow MSCs [ 17 ], the Hoffa's fat pad offers a number of practical advantages, including familiarity and accessibility for the orthopaedic surgeon.
In addition, the potential yield of cells is greater compared with BM by virtue of the fat pad size. There is considerable interest in synovium as another source of MSCs for OA cellular therapy development [ 11 ], but it is a very heterogeneous in OA. It may vary in thickness, show fibrotic or haemorrhagic changes or inflammation, the latter of which in itself may be detrimental to stem cell function [ 38 ]. In comparison to knee joint synovitis, which is common and variable in extent in OA, the recognition of inflammatory changes in Hoffa's fat pad is less frequent [ 38 ].
This factor, the consistent location, size and accessibility of the fat pad could make it a good source for cell therapy development in OA. The present study also provides some intriguing immunophenotypic data as to the identification of the fat pad MSC in vivo. Studies are ongoing to prospectively isolate LNGFR positive cells in the fat pad and to determine whether they are enriched for clonogenic and chondrogenic cellular fractions in numbers sufficient to be used directly as cell therapy without prior culture expansion.
Besides the optimal source of repair cells, the other important issue remains an ability to predict a chondrogenic outcome from a given cell population. Cell surface markers represent the easiest solution, as flow cytometry-based assays are reproducible, automated and highly objective.
In relation to chondrogenesis-related markers, FGF receptor 3, BMP2 and Col2A1 were proposed in the past [ 40 ], but no well-characterized commercial antibodies are yet available to monitor these markers at the protein level. However, most researchers consider it as a common MSC marker, and this was confirmed in our previous work [ 18 , 33 ]. Similarly, the expression of other putative chondro-predictive markers, namely CD44 and CD [ 31 ], did not differ in the tissues tested in this study, nor did it reflect the loss of chondrogenic capacity following extended passaging of chondrocytes.
In conclusion, this study showed that cells that meet the full criteria for MSCs, namely clonogenicity and multipotentiality were absent in OA cartilage, irrespective of the source, but could be readily isolated from Hoffa's fat pads. The Hoffa's fat pad may therefore represent a good source for autologous MSCs and our findings may facilitate the exploration of stem cell therapy in older subjects with OA.
Further work is needed to establish robust molecular markers predictive of its chondrogenicity in vitro and in vivo and to prospectively purify the fat pad MSC population. Further studies are also needed to determine whether the observed lack of MSC activity in articular cartilage may be a contributory factor to disease progression in OA. We wish to thank the staff in the orthopedic theatre at The Calderdale Royal Hospital, Halifax for their help in collecting samples and in particular Julie Madden for organizing transport between the hospitals, and Elizabeth Hensor for her statistical advice.
Oxford University Press is a department of the University of Oxford. It furthers the University's objective of excellence in research, scholarship, and education by publishing worldwide. Sign In or Create an Account. Sign In. Advanced Search. Article Navigation. Close mobile search navigation Article Navigation. Volume Article Contents. Materials and methods. A comparative assessment of cartilage and joint fat pad as a potential source of cells for autologous therapy development in knee osteoarthritis A.
Oxford Academic. Google Scholar. Cite Citation. Permissions Icon Permissions. Abstract Objectives. Mesenchymal Stem Cells , Osteoarthritis , Cartilage. Open in new tab Download slide. T able 1. Open in new tab. Mesenchymal stem cells: clinical applications and biological characterization. Search ADS. Stem cells for repair of cartilage and bone: the next challenge in osteoarthritis and rheumatoid arthritis.
Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation. Plasticity of clonal populations of dedifferentiated adult human articular chondrocytes.
Interspecific Differences in the Macro and Microscopic Structure of Articular Cartilage
Dedifferentiated adult articular chondrocytes: a population of human multipotent primitive cells. Stemming cartilage degeneration: adult mesenchymal stem cells as a cell source for articular cartilage tissue engineering. De Bari. Distinct biological properties of human mesenchymal stem cells from different sources.
Comparison of human stem cells derived from various mesenchymal tissues. Superiority of synovium as a cell source. Lessons from musculoskeletal stem cell research: the key to successful regenerative medicine development. PGE2 levels are increased to an impact load on articular cartilage or during cartilage degeneration. Unstimulated human chondrocytes do not contain detectable COX Prostaglandins, whose synthesis involves COX-1, are responsible for maintenance and protection of the gastrointestinal tract, while prostaglandins, whose synthesis involves COX-2, are responsible for inflammation and pain.
One of the main prostaglandins involved in this inflammatory reaction is PGE2. Since normal articular chondrocytes produce very little PGE2 and osteoarthritic chondrocytes produce a lot of it through the COX-2 enzyme, it would make sense from a traditional medical point of view to attack arthritis pain from this angle. The articular chondrocytes make PGE2 in response to injury to stimulate healing. Osteoarthritic cartilage spontaneously releases PGE2 in levels at least fold higher than normal cartilage and fold higher than normal cartilage stimulated with cytokines and endotoxin.
The long-term consequences, of course can be an acceleration of the degenerative osteoarthritic process. Cranial acetabular migration, a measure of acetabular destruction, was present in 37 hips and absent in Those patients with serious hip destruction when compared with those who did not have the acetabular destruction did not differ in sex, age, pain grading, or walking ability. According to the researchers, NSAID use was associated with progressive formation of multiple small acetabular and femoral subcortical cysts and subchondral bone thinning. Researchers in Norway studied the course of osteoarthritis in hips of patients with radiographs over a three year period.
Specifically the researchers found that in the three year period of the study, the osteoarthritic hips treated with the NSAID had more cysts, altered bone structure, and overall hip destruction. He performed further investigations on the extirpated cut out from surgery femoral heads with examination of cut surface, slab radiographs, and histology. Many of the heads, and especially those with changes attributable to NSAIDs, were found to have microscopic fragmentation of the bony trabeculae giving the appearance of a jammed marrow space.
They did a large study to compare the rate of radiographic progression in knee osteoarthritis comparing indomethacin NSAID with placebo. The study involved 20 rheumatology clinics in the United Kingdom. Patients received indomethacin 25mg three times daily or a placebo. The average person in each group was around 60 years of age and had osteoarthritis in the knee for over five years. The study involved 85 clients in the indomethacin group and 85 in the placebo group. Radiographic analysis was done yearly and the radiographic grade was judged by two observers using a six point scale.
The average length of follow-up was three years. By the third year of the study, the results were so dramatic demonstrating the acceleration of the degeneration of the articular cartilage in the knee osteoarthritic patient treated with indomethacin that this part of the study had to be stopped. There were more than twice as many patients showing deterioration in the indomethacin group as the placebo. The authors noted that the risk of deterioration within a one year period in patients taking indomethacin relative to placebo was 2.
If NSAIDs, by inhibiting pain and inflammation in osteoarthritic joints, cause people with OA to overuse a damaged joint, this should result in accelerating joint degeneration and joint replacements at an earlier time or, alternatively, if treatment with NSAIDs alters cartilage metabolism and inhibits joint healing, an acceleration of articular cartilage degeneration should be seen.
Numerous studies have shown that non-steroidal anti-inflammatory drugs, particularly indomethacin, increase the rate of progression of osteoarthritis of the hip and knee. In a recent landmark study, Dutch researchers studied more than subjects with hip OA and with knee OA. The researchers evaluated radiographs of the hip and knee at baseline and follow-up. The mean follow-up period was 6. They found that long-term use of the NSAID diclofenac was associated with a more than twofold increase in radiologic progression of hip osteoarthritis and a threefold increase in progression observed in the knee.
The authors noted that this may have resulted in an underestimation of the reported associations. Whether this occurs because of a true deleterious effect on cartilage or because of excessive mechanical loading on a hip or knee following pain relief, remains to be investigated. It is important to remember that pain has a physiologic function: if a joint produces pain when it is used, it is a signal for the body to use that joint less or else the structure eliciting the pain will be further damaged.
One study focused on a group of patients with hip osteoarthritis who needed to have a joint replacement in the not-too-distant future. Over the next months, the patients were asked about their joint pain, and radiographs of their hips were taken. The patients given the NSAID had more progression of their hip radiographs and needed to have joint replacements performed in half the time as the group given acetaminophen.
The authors speculated why this occurred. They noted that the NSAID might have prevented normal cartilage turnover and repair, and accelerated the joint degeneration; or, more likely, the potent medication decreased joint pain and those subjects were therefore more active.
This latter notion has actually been studied: patients who take NSAIDs for knee OA put increased joint forces on their knees with walking because of pain relief, compared to those who do not have pain relief taking nothing, or just a placebo. Other researchers have also confirmed that NSAID users need total joint replacements sooner than those who do not take them. It is clear from the scientific literature that NSAIDs from in vitro and in vivo studies in both animals and humans have a significant negative effect on cartilage matrix which causes an acceleration of the deterioration of articular cartilage in osteoarthritic joints.
The preponderance of evidence shows that NSAIDs have no beneficial effect on articular cartilage and accelerate the very disease for which they are most used and prescribed. While the rapid deterioration of joints after long-term NSAID treatment can be from a loss of proactive pain sensations, it is much more likely that it is a direct effect of NSAIDs on cartilage. Clinically this is manifested as an accelerated progression of the knee or hip osteoarthritis as seen by standard radiographs. The long-term consequence of the deterioration of the joint is a need for joint replacement. This author notes that massive NSAID use in osteoarthritic patients since their introduction over the past forty years is one of the main causes of the rapid rise in the need for hip and knee replacements both now and in the near future.
Various scientific papers and consensus groups have stated that there is no convincing data to show that the widely used NSAIDs and recommended selective COX-2 inhibitors have favorable effects on cartilage. Specifically the recommendation was as follows: In patients with symptomatic hip or knee osteoarthritis, non-steroidal anti-inflammatory drugs NSAIDs should be used at the lowest effective dose but their long-term use should be avoided if possible.
Other groups have raised similar sentiments. The committees of the International League Against Rheumatism and the World Health Organization came up with guidelines for the testing of new drugs in osteoarthritis. The consensus from these meetings resulted in recommendations by The European Group for the Respect of Ethics and Excellence in Science GREES for governmental registration and approval of new drugs used in the treatment of OA and have added the requirement that the drug not have a deleterious effect on the diseased and non-diseased contra lateral joint; i.
While it is admirable for the various consensus and rheumatology organizations to educate doctors and the lay public about the necessity to limit NSAID use in OA, this author RH feels the warnings are not enough. Within the last year, for instance, the FDA has again implemented new rules requiring stronger and more extensive label warnings in addition to the heart disease risks regarding the risk of liver damage and stomach bleeding for people taking common over-the-counter pain relievers.
The chance is higher if you are age 60 or older, have had stomach ulcers or bleeding problems, take a blood thinning or steroid drug, take other drugs containing prescription or nonprescription NSAIDs, have three or more alcoholic drinks every day using this product, take more or for a longer time than directed.
The lay public for whom NSAIDs are prescribed and recommended by both health care professionals and drug manufacturers should be aware that long-term NSAID use is detrimental to articular cartilage. Physicians, allied health care professionals, and drug manufacturers should be required to inform the lay public that NSAID use can accelerate OA articular cartilage degeneration.
A strict warning label on these medications should read as follows:. If this does not occur, then most likely the exponential rise in degenerative arthritis and subsequent musculoskeletal surgeries, including knee and hip replacements, as well as spine surgeries, will continue for decades to come. Skip to content. Hauser, MD. While it is admirable for the various consensus and rheumatology organizations to educate doctors and the lay public about the necessity to limit NSAID use in OA, the author recommends that the following warning label be on each NSAID bottle: The use of this nonsteroidal anti-inflammatory medication has been shown in scientific studies to accelerate the articular cartilage breakdown in osteoarthritis.
Figure 1. Chondrocytes — the only cells in cartilage tissue responsible for the synthesis of collagen and proteoglycans that makeup the cartilage matrix. However, privileging on the composition, the generation of a biphasic scaffold with a longitudinal roughness of the inner channels that serves as a guide for the correct adhesion of the cells; it results in a topography that truly emulates the osteochondral unit and shows in vivo a superior regeneration of the osteochondral tissue compared to the random structure [ 55 ].
Therefore, not only the pore size and porosity should be taken into account for a correct design, but also the alignment of the channels within the scaffold influencing cell migration and the proper pattern fibers of the ECM. Likewise, multiphase can be assembled on the basis of a single biomaterial. It is possible to modify the physical properties such as roughness, pore size, and interconnectivity particularizing according to the phase, selecting a specific type of porogen and particle size, as well as through the use of different solvents and the polymerization process.
The conformation of this design showed not only the ability to promote the differentiation of human mesenchymal stem cells toward the chondrogenic or osteogenic lineage, but also in addition, by having a well-stratified biphasic structure, the loading behavior validated the compression properties. By the same token, a single biomaterial can be used and can generate distinct microenvironment using different molecules to biofunctionalize in a tissue-specific manner.
Certainly, no biomaterial is intrinsically capable of satisfying all the requirements for the manufacture of complex and stratified tissues, so the biofunctionalization of these is presented as an alternative procedure to adapt the properties of the biomaterials to the needs of the chondral or bone tissue. The final structure leads to the formation of channel-like, parallel aligned pores. In order to generate a chondral environment, alginate is biofunctionalized with hyaluronic acid, while for the bone phase, hydroxyapatite is used.
This simple procedure generates two well-defined layers characterized by different microstructure and mechanical properties, which provide a suitable environment for cells to form the respective tissue. Although an interface between the chondral and bone areas of the implant is not structured, a stable connection between them is clearly demonstrated, which positively impacts the mechanical properties in the final design.
According to the influence of biofunctionalization, it was demonstrated by gene expression analysis that the embedded stem cells differentiated into the chondrogenic lineage when they were cultivated in chondrogenic medium; additionally, under the stimulation of the hyaluronic acid present in this phase, the chondrocyte phenotype remained stable. Biofunctionalization, especially for monolithic scaffolds, is a useful alternative to provide chondro- and osteoinductive properties.
Aragonite is a biomaterial from coral exoskeletons, similar to human bone including its 3D structure and pore interconnections as well as its crystalline form of calcium carbonate CaCO 3 [ 58 ]. That features confers improved osteoconductive ability, suitable for bone regeneration. Interestingly, specific coral species differ in size and interconnectivity of coral pores, which expands the range of applications for different tissues.
We have already discussed before, the relevance of channel generation aligned in parallel to guide the adhesion of the cells in the chondral phase and the subsequent structuring of the ECM. In this design, in addition to the biofunctionalization with hyaluronic acid, the mechanical modification of drilled channels is also added. The combination of the two strategies showed in a model of joint damage in goat the best results compared to aragonite alone, and in the absence of parallel channels; it means a cartilaginous repair tissue with hyaline cartilage as shown by the marked expression of proteoglycans, as well as of collagen type II and absence of collagen type I.
Vascularization is the bottleneck in tissue engineering. Creating constructs in the laboratory that lack of the proper vessels network will fail after implantation as the cells will not get enough oxygen and nutrients and will die. This fact is even more significant for osteochondral regeneration. Bone is a highly vascularized tissue while cartilage is avascular. When vascular networks invade cartilage surface from the subchondral zone, it might lead to an ossification of the cartilage from the deep and intermediate zone implying a joint damage and increasing pain.
The design of the optimal scaffold to control angiogenesis, promoting vessel growth from preexisting ones, on the bone side and inhibiting it on the cartilage side is relevant for osteochondral regeneration. One strategy to improve bone formation is to use growth factors GFs that can activate angiogenesis within the scaffold.
Uploading VEGF is widely used, as the VEGF activates endothelial precursor cell EPC migration and proliferation activating the angiogenic process, and subsequently promotes the recruitment and survival of bone forming cells improving bone regeneration. However, the presence of high levels of VEGF is one of the factors related to OA progression, inducing cartilage degeneration and pain [ 61 ].
Therefore, the scaffold design for osteochondral regeneration must fulfill different properties that are shared by the two tissues, such as cell adhesion and proliferation and a high production of ECM; but others must deal with angiogenic promotion for bone or angiogenic inhibition for cartilage. Furthermore, the already observed side effects of supraphysiological doses of bone-related GFs heterotopic bone growth, pseudoarthrosis, local inflammation, and immune response [ 62 ] must be controlled by means of delivery vehicles that will ensure the bioactivity of these molecules and the remaining in the target location over the therapeutic timeframe.
The Basic Science of Articular Cartilage
This can be done by covalent attachment to the scaffold, noncovalent binding, or with the nanoparticle carriers. The use of this synthetic polymer completely inhibited angiogenesis by the interaction of the sulfonic acid groups with the bFGF and VEGF modulating their activity in the processes of endothelial cell migration and proliferation. Thus, the fabrication of a biphasic scaffold by combining two different polymers that can control angiogenesis can be an efficient approach.
An innovative approach that has been tested recently is the use of microRNAs miRNAs to modulate cell activity for regenerative medicine applications. The roles of these miRNAs on bone diseases such as osteoporosis, osteoarthritis, and rheumatoid arthritis have been recently highlighted. The use of these molecules as miRNA regulators can be done by using synthetic molecules, which either mimic or repress the function of endogenous miRNAs. The mimicking molecules will enhance the suppression of the target protein synthesis by degrading the miRNA or inhibiting the protein translation.
On the other hand, miRNAs inhibitors antagomirs preventing the activity inside the cells will lead to a rise of mRNA and protein expression. This approach can be used to upload scaffolds with either agonist or antagonist molecules to induce or avoid vascularization. Many scaffolds have been designed to fulfill the function of miRNA delivery, mainly hydrogels, nanofibers, and porous or spongy scaffolds. Besides, the normal desired properties such as biocompatibility, easy fabrication, easy sterilization, proper mechanical properties, and adequate porosity for vessels growth, the material must retain the miRNA complexes while facilitating their sufficient exposure to the infiltrating cells without affecting its mechanical properties [ 68 ].
Biomaterial scaffold properties are fundamental to guide and recreate the native environment. The biomaterials for osteochondral applications in first insight must be biocompatible and should be intrinsically osteoinductive, osteoconductive, chondroinductive, or chondroconductive, and not less insignificant, and must possess a degradation rate that allows the formation of new tissue. As previously stated, an ideal scaffold for the treatment from a multiphase point of view must have a chondrogenic matrix that is flexible, resistant and with pores small enough to mimic the hyaline cartilaginous matrix and an osteogenic matrix that should be mechanically competent similar to cancellous bone and bioactive, which has larger pores that mimic the microenvironment of the subchondral bone.
Achieving an articular cartilage design capable of mimicking its anisotropic mechanical behavior, still represents one of the greatest challenges in the cartilage tissues engineering [ 69 ]. In addition, the ideal biomaterial for cartilage should allow the cartilage composition to be recreated in terms of the liquid and solid phases of the connective tissue, reproduce its zonal organization, and facilitate the integration of the neoformed tissue with the adjacent native tissue.
Functionally, we can classify biomaterials into: protein-based polymers, such as fibrin, gelatin, collagen, and silk fibroin [ 70 , 71 , 72 , 73 , 74 ]. Biopolymers based on carbohydrates such as alginate, chitosan, agarose and polyethylene glycol [ 75 , 76 , 77 , 78 ], and synthetic polymers such as polylactic acid, polyglycolic acid, polycaprolactone and polylactic-coglycolicacid PLGA are the most common [ 79 , 80 , 81 ]. These kinds of biomaterials are comprised of cross-linked polymers that swallow a great amount of water, which empathizes with the features of cartilage ECM, thus favoring the maintenance of spherical morphology within the scaffold [ 76 ].
Furthermore, synthetic materials and growth factors can be added in order to enhance chondrogenesis. A material with adequate characteristics for cartilage engineering is chitosan, a polycationic polysaccharide that can be degraded enzymatically by the lysozyme present in the MEC of human cartilage. Chitosan has a chemical similarity with GAGs, which gives it the ability to interact with them [ 82 ]; through various in vitro studies, it has been demonstrated that scaffolds based on chitosan especially in combination with other biomaterials such as collagen II [ ], hyaluronic acid [ 83 ], or fibroin [ 84 ] promote chondrogenic activity and support the production of aggrecan and type II collagen, thus improving cartilage repair [ ].
Among the materials of a protein nature is gelatin, which is formed from denatured collagen and can bind to growth factors, proteins, and peptides and is also capable of promoting efficient cell adhesion.
On the other hand, there is the collagen that constitutes the main structural component of the ECM, and its use as a scaffolding material allows the cells to retain their phenotypes [ 85 ]. Collagen is a naturally occurring protein found in various fibrous tissues such as bone and cartilage.
Collagen-based scaffolds have been used for cartilage tissue engineering applications as biomaterials due to its biocompatibility and biodegradability.
Treatment of Articular Cartilage Defects: Focus on Tissue Engineering
Type I collagen gels seeded with bone marrow-derived MSCs have shown the formation of cartilage and subchondral bone after implantation in a full-thickness osteochondral defects macaque model. After 24 weeks, the defect had been covered with cartilage-rich reparative tissue, suitable integration with the surrounding cartilage tissue, and restoration of trabecular subchondral bone [ 86 ]. As part of this group of biomaterials is the silkworm fibroin, which is a natural biopolymer, with properties such as biocompatibility and biodegradability that allow it to be currently used for the development of a wide variety of biomedical devices and new regeneration technologies [ 87 ].
In this context, silk fibroin has interesting applications in the engineering of hard and soft tissues and has diverse characteristics among which is included the ability to support the proliferation and differentiation of various cell types, making it an attractive therapeutic candidate in cartilage regenerative medicine Table 1 [ 56 , ].
Silk fibroin has been used in several medical applications, and it can be used as fiber [ 92 ], electrospun fibers [ 93 ], films [ 94 ], or hydrogels [ 95 ]. The versatility of fibroin as a biomaterial makes it suitable for any type of application in tissue engineering, and applications that demonstrate greater maturity and close to its final application are in the field of regeneration of bone, cartilage, and ligaments.
In this regard, a very interesting application is the reconstruction of the cruciate ligament of the knee through the elaboration of a cord of silk fibers that later are sown with mesenchymal cells of the bone marrow that differentiate to ligament tissue, offering a mechanical resistance much superior to that of other organic materials and a great biocompatibility. This application is already in commercial phase in the United States, by a company specialized in the development of biomaterials based on silk fibroin Serica [ 96 ].
Regarding the regeneration of cartilage, fibroin has been used for the manufacture of biphasic implants in combination with bioactive ceramics or 70S bioactive glass, which has allowed the obtaining of scaffolds with stratified properties capable of satisfying the complex and diverse regenerative requirements of the osteochondral tissue [ 97 ]. Several biodegradable and biocompatible polymers of synthetic origin have been developed for biomedical applications. In vivo studies have shown that highly crystalline PLLA degrades completely in 5 years, while mostly amorphous PDLLA loses strength in less than 2 months and is degraded in 1 year [ ].
The material properties, degradation rates, and tissue compatibility of PLA can be modified by copolymerization with other monomers, resulting in copolymers such as poly lactic acid-co-caprolactone PLGA , poly lactic acid-co-caprolactone PLCL , poly lactic acid-co-ethylene glycol PLEG , and poly lactic acid-co-glutamic acid PLGM ; this makes them biomaterials with highly adaptable properties for broad biomedical applications Table 1 [ , , , , ].
PGA has demonstrated good chondrogenesis both in vitro and in vivo [ ]. A combination of a cell-free poly L-lactic-coglycolic acid scaffold and in situ bone marrow stem cells has been used for focal full-thickness cartilage defects in a rabbit model, demonstrating suitable integration of the implant and hyaline-like cartilage regeneration in 24 weeks [ ]. This scaffold is cellularized with autologous articular chondrocytes showing improved clinical scores in human trials [ ].
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The restoration of osteochondral tissue damage should be focused on the physiological features and the structure of the tissues that make it up cartilage and bone , considering the different microenvironments that coexist in the native tissue. Through tissue engineering, multiphase designs have been developed, such as those discussed throughout this chapter that aspire to achieve this goal.
Although there are few multiphase designs that are currently available for a clinical application, they open an important direction for the rigorous evaluation of the designs found on this path. For clinical trials, only one clinical case has been reported in a year-old man with an injury Outerbridge grade IV. The lesion was treated successfully and resumed normal activity after 18 months.
In a follow-up at 24 months, restoration of the articular surface was demonstrated by MRI [ ]. Although the results were encouraging, the occupation of the patient athlete could have a positive influence on the observed result, this makes it necessary to develop clinical trials in a larger number of patients under controlled conditions in order to extrapolate the benefits to a wider segment of the population.
The scaffold was used at first by direct implantation for the treatment of focal articular surface defects, but it showed some controversial results [ ]. Several clinical studies have described a slow chondral restoration in the area of the lesion, due to poor bone repair [ ], together with the poor integration of the implant with the surrounding tissue [ ]. The long-term follow-up up to 2 years have also been controversial; however, the constant was delayed formation of the subchondral lamina [ ].
By preclinical tests on sheep and horses, it was possible to demonstrate the safety of the implant, but also that allowed the regeneration of the type II collagen-rich tissue after 6 months; this is a cell-free design [ , ]. Throughout several clinical trials developed in such diverse populations ranging from 28 to 60 years and with a lesion size ranging from 1. The evolution of the regeneration process has demonstrated the formation of subchondral bone and maturation of the chondral tissue in a period of 6 months. The evaluation by a high-resolution magnetic resonance imaging MRI shows the complete repair of the tissue in a period of 2 years in A cartilage repair treatment using tissue engineering comprises the implantation of bioabsorbable scaffolds that at first fill a chondral or osteochondral defect, then the production of cartilage repair tissue depends on the de novo synthesis of cartilage matrix elements.
Such scaffolds support the local migration of cells chondrogenic or osteogenic that basically synthesize new extracellular matrix. The aim of all cartilage replacement strategies should focus on reconstruction of hyaline cartilage with its hierarchical organization; however, most of the current strategies based on monophasic designs lead to the production of fibrocartilage, which has inferior biological and mechanical characteristics compared to hyaline cartilage.
The design of multiphasic scaffolds aims at congruence with that of hierarchical nature, and from the studies that have been carried out over the past few years, it is clear that as a consequence, it substantially improves the integration of the implant with the surrounding osteochondral tissue, and positively influences the functional regeneration of both chondral and bone tissues.
The use of scaffolding in order to recapitulate as much as possible the hierarchical structure seems to be not enough. The decision to cellularize or maintain a cell-free scaffold is crucial, and the answer will depend on the 3D system in a particular way; therefore, cellularization in each of the chondral and bone phases must be taken into account for the final design.
The needed to mimic the ECM on a molecular level is another main goal that demands to be taken into account, so the bioactivation of the biomaterials with elements such as synthetic materials as the ceramics tricalcium phosphate, hydroxyapatite, and bioactive glass , or even the same decellularized tissue matrix, turns out to be a valuable tool for cartilage design, since these materials enhance the growth of a bone-like layer to support the overlying cartilage to the existing osteochondral defect. Experimental studies are ongoing to evaluate innovative multiphase designs regarding the interaction with cells and the environment in an in vivo framework.
In vivo trials using small animal models provide innovative concepts in osteochondral tissue engineering; nonetheless, to reach the development of clinical trials in humans, it is important to follow successful experiments using animal models that have loads and joint dimensions similar to humans. Animals such as sheep, pigs, and horses have surgically created defect sizes ranging from 0. The body weights of these animals are also comparable or much heavier than humans, which makes them more appropriate models to predict the results in clinical trials.
Although the challenge to incorporate the use of multiphase designs to the clinic is still great, from the results observed in the wide range of studies, it is possible to conclude that tissue engineering approaches based on multiphasic scaffolds represent a promising therapeutic treatment for the regeneration of osteochondral defects. Moreover, based on the clinical results, it seems that a three-phase approach offers the most promising results with patients. Special thanks to Stefano Serati for his valuable support and inspiration for the completion of this chapter. Licensee IntechOpen.