Infrapatellar Fat Pad-derived Stem Cells Research Articles

Injectable hyperbranched PEG crosslinked hyaluronan hydrogel microparticles containing mir-99a-3p modified subcutaneous ADSCs-derived exosomes was beneficial for long-term treatment of osteoarthritis

Exosomes (Exos) secreted by adipose-derived stem cells (ADSCs) have shown potential in alleviating osteoarthritis (OA). Previous studies indicated that infrapatellar fat pad (IPFP) derived stem cells (IPFSCs) may be more suitable for the treatment of OA than subcutaneous adipose tissue (ScAT) derived stem cells (ScASCs). However, it remains unclear which type of Exos offers superior therapeutic benefit for OA. This study first compared the differences between Exos derived from IPFP stem cells (ExosIPFP) and ScAT stem cells (ExosScAT) in OA treatment. Results suggested that ExosIPFP significantly inhibit the degradation of cartilage extracellular matrix (ECM) than ExosScAT, following this, the differences in microRNA (miRNA) expression between the two types of Exos using small RNA sequencing were performed. Subsequently, miR-99 b-3p was chosen and over-expressed in ExosScAT (ExosScAT−99b−3p), both in vivo and in vitro experiments demonstrated its efficacy in inhibiting the expression of ADAMTS4, promoting the repair of the ECM in OA. Finally, microfluidic technology was performed to fabricate a hyaluronan-based hydrogel microparticles (HMPs) for encapsulating Exos (HMPs@exos), the injectability, sustained release of Exos and long-term therapeutic effect on OA were validated. In summary, these results suggest miR-99 b-3p regulates the degradation of cartilage ECM by targeting ADAMTS4, the upregulation of miR-99 b-3p in ExosScAT would enable them to exhibit comparable or even superior effectiveness to ExosIPFP for OA treatment, making it a promising approach for OA treatment. Considering the abundant resources of ScAT and the limited availability of IPFP, ScAT harvested through liposuction could be genetically engineered to yield Exos for OA treatment. Furthermore, the encapsulation of Exos in HMPs provides an injectable sustained local drug release system, which could potentially enhance the efficacy of Exos and hold potential as future therapeutic strategies.

Matrix from urine stem cells boosts tissue-specific stem cell mediated functional cartilage reconstruction.

Articular cartilage has a limited capacity to self-heal once damaged. Tissue-specific stem cells are a solution for cartilage regeneration; however, ex vivo expansion resulting in cell senescence remains a challenge as a large quantity of high-quality tissue-specific stem cells are needed for cartilage regeneration. Our previous report demonstrated that decellularized extracellular matrix (dECM) deposited by human synovium-derived stem cells (SDSCs), adipose-derived stem cells (ADSCs), urine-derived stem cells (UDSCs), or dermal fibroblasts (DFs) provided an ex vivo solution to rejuvenate human SDSCs in proliferation and chondrogenic potential, particularly for dECM deposited by UDSCs. To make the cell-derived dECM (C-dECM) approach applicable clinically, in this study, we evaluated ex vivo rejuvenation of rabbit infrapatellar fat pad-derived stem cells (IPFSCs), an easily accessible alternative for SDSCs, by the abovementioned C-dECMs, in vivo application for functional cartilage repair in a rabbit osteochondral defect model, and potential cellular and molecular mechanisms underlying this rejuvenation. We found that C-dECM rejuvenation promoted rabbit IPFSCs' cartilage engineering and functional regeneration in both ex vivo and in vivo models, particularly for the dECM deposited by UDSCs, which was further confirmed by proteomics data. RNA-Seq analysis indicated that both mesenchymal-epithelial transition (MET) and inflammation-mediated macrophage activation and polarization are potentially involved in the C-dECM-mediated promotion of IPFSCs' chondrogenic capacity, which needs further investigation.

Restoring Osteochondral Defects through the Differentiation Potential of Cartilage Stem/Progenitor Cells Cultivated on Porous Scaffolds.

Cartilage stem/progenitor cells (CSPCs) are cartilage-specific, multipotent progenitor cells residing in articular cartilage. In this study, we investigated the characteristics and potential of human CSPCs combined with poly(lactic-co-glycolic acid) (PLGA) scaffolds to induce osteochondral regeneration in rabbit knees. We isolated CSPCs from human adult articular cartilage undergoing total knee replacement (TKR) surgery. We characterized CSPCs and compared them with infrapatellar fat pad-derived stem cells (IFPs) in a colony formation assay and by multilineage differentiation analysis in vitro. We further evaluated the osteochondral regeneration of the CSPC-loaded PLGA scaffold during osteochondral defect repair in rabbits. The characteristics of CSPCs were similar to those of mesenchymal stem cells (MSCs) and exhibited chondrogenic and osteogenic phenotypes without chemical induction. For in vivo analysis, CSPC-loaded PLGA scaffolds produced a hyaline-like cartilaginous tissue, which showed good integration with the host tissue and subchondral bone. Furthermore, CSPCs migrated in response to injury to promote subchondral bone regeneration. Overall, we demonstrated that CSPCs can promote osteochondral regeneration. A monophasic approach of using diseased CSPCs combined with a PLGA scaffold may be beneficial for repairing complex tissues, such as osteochondral tissue.

Stem cells immortalized by hTERT perform differently from those immortalized by SV40LT in proliferation, differentiation, and reconstruction of matrix microenvironment

Although matrix microenvironment has the potential to improve expanded stem cell proliferation and differentiation capacity, decellularized extracellular matrix (dECM) deposited by senescent cells does not contribute to the rejuvenation of adult stem cells, which has become a barrier to personalized stem cell therapy. Genetic modification is an effective strategy to protect cells from senescence but it carries the increased risk of malignant transformation and genetic instability. In this study, lentivirus carrying either human telomerase reverse transcriptase (hTERT) or simian virus 40 large T antigen (SV40LT) was used to transduce human infrapatellar fat pad-derived stem cells (IPFSCs). We found that virus transduction modified the proliferative, chondrogenic, and adipogenic abilities of IPFSCs. Interestingly, dECM deposited by immortalized cells significantly influenced replicative senescent IPFSCs in proliferation and differentiation preference, the effect of which is hinged on the approach of immortalization using either SV40LT or hTERT. Our findings indicate both dECM expansion and immortalization strategies can be used for replicative senescent adult stem cells’ proliferation and lineage-specific differentiation, which benefits future stem cell-based tissue regeneration. This approach may also work for adult stem cells with premature senescence in elderly/aged patients, which needs further investigation. Statement of significanceAdult stem cells are a promising solution for autologous cell-based therapy. Unfortunately, cell senescence due to donor age and/or ex vivo expansion prevents clinical application. Recent progress with decellularized extracellular matrix provides a potential for the rejuvenation of senescent stem cells by improving their proliferation and differentiation capacities. Given the fact that the young matrix can provide a healthy and energetic microenvironment, in this study, two approaches using lentivirus transduction of hTERT and SV40LT were compared. The goal was to immortalize donor cells for deposition of decellularized extracellular matrix. The matrix was demonstrated to contribute diverging effects on the chondrogenic and adipogenic differentiation of expanded stem cells and exhibited proliferation benefits as well. These findings provide an invaluable asset for stem cell-based tissue regeneration.

Unfavorable Contribution of a Tissue-Engineering Cartilage Graft to Osteochondral Defect Repair in Young Rabbits.

A stem cell-based tissue-engineering approach is a promising strategy for treatment of cartilage defects. However, there are conflicting data in the feasibility of using this approach in young recipients. A young rabbit model with an average age of 7.7 months old was used to evaluate the effect of a tissue-engineering approach on the treatment of osteochondral defects. Following in vitro evaluation of proliferation and chondrogenic capacity of infrapatellar fat pad-derived stem cells (IPFSCs) after expansion on either tissue culture plastic (TCP) or decellularized extracellular matrix (dECM), a premature tissue construct engineered from pretreated IPFSCs was used to repair osteochondral defects in young rabbits. We found that dECM expanded IPFSCs exhibited higher proliferation and chondrogenic differentiation compared to TCP expanded cells in both pellet and tissue construct culture systems. Six weeks after creation of bilateral osteochondral defects in the femoral trochlear groove of rabbits, the Empty group (left untreated) had the best cartilage resurfacing with the highest score in Modified O’Driscoll Scale (MODS) than the other groups; however, this score had no significant difference compared to that of 15-week samples, indicating that young rabbits stop growing cartilage once they reach 9 months old. Interestingly, implantation of premature tissue constructs from both dECM and TCP groups exhibited significantly improved cartilage repair at 15 weeks compared to those at six weeks (about 9 months old), indicating that a tissue-engineering approach is able to repair adult cartilage defects. We also found that implanted pre-labeled cells in premature tissue constructs were undetectable in resurfaced cartilage at both time points. This study suggests that young rabbits (less than 9 months old) might respond differently to the classical tissue-engineering approach that is considered as a potential treatment for cartilage defects in adult rabbits.

Impact of Fibronectin Knockout on Proliferation and Differentiation of Human Infrapatellar Fat Pad-Derived Stem Cells.

Fibronectin plays an essential role in tissue development and regeneration. However, the effects of fibronectin knockout (FN1-KO) on stem cells' proliferation and differentiation remain unknown. In this study, CRISPR/Cas9 generated FN1-KO in human infrapatellar fat pad-derived stem cells (IPFSCs) was evaluated for proliferation ability including cell cycle and surface markers as well as stemness gene expression and for differentiation capacity including chondrogenic and adipogenic differentiation. High passage IPFSCs were also evaluated for proliferation and differentiation capacity after expansion on decellularized ECM (dECM) deposited by FN1-KO cells. Successful FN1-KO in IPFSCs was confirmed by Sanger sequencing and Inference of CRISPR Edits analysis (ICE) as well as immunostaining for fibronectin expression. Compared to the GFP control, FN1-KO cells showed an increase in cell growth, percentage of cells in the S and G2 phases, and CD105 and CD146 expression but a decrease in expression of stemness markers CD73, CD90, SSEA4, and mesenchymal condensation marker CDH2 gene. FN1-KO decreased both chondrogenic and adipogenic differentiation capacity. Interestingly, IPFSCs grown on dECMs deposited by FN1-KO cells exhibited a decrease in cell proliferation along with a decline in CDH2 expression. After induction, IPFSCs plated on dECMs deposited by FN1-KO cells also displayed decreased expression of both chondrogenic and adipogenic capacity. We concluded that FN1-KO increased human IPFSCs' proliferation capacity; however, this capacity was reversed after expansion on dECM deposited by FN1-KO cells. Significance of fibronectin in chondrogenic and adipogenic differentiation was demonstrated in both FN1-KO IPFSCs and FN(–) matrix microenvironment.

Effect of passaging on the stemness of infrapatellar fat pad‑derived stem cells and potential role of nucleostemin as a prognostic marker of impaired stemness.

Infrapatellar fat pad-derived stem cells (IFPSCs) are emerging as an alternative to adipose tissue-derived stem cells (ADSCs) from other sources. They are a reliable source of autologous stem cells obtained from medical waste that are suitable for use in cell-based therapy, tissue engineering and regenerative medicine. Such clinical applications require a vast number of high-quality IFPSCs. Unlike embryonic stem cells (ESCs), ADSCs and IFPSCs have limited population doubling capacity; however, in vitro expansion of primary IFPSCs through multiple passages (referred to as P) is a crucial step to acquire the desired population of cells. The present study investigated the effect of multiple passages on the stemness of IFPSCs during expansion and the possibility of predicting the loss of stemness using certain markers. IFPSCs were isolated from infrapatellar fat pad tissue resected during knee arthroplasty performed on aged patients (>65 years old). These cells from the stromal vascular fraction were serially passaged to at least to P7, and their stemness characteristics were examined at each passage. It was observed that IFPSCs maintained their spindle-shaped morphology, self-renewability and homogeneity at P2-4. Furthermore, immunostaining revealed that these cells expressed mesenchymal stem cell (CD166, CD90 and CD105) and ESC markers [Sox2, Nanog, Oct4 and nucleostemin (NS)], whereas the hematopoietic stem cell marker CD45 was absent. These cells were also able to differentiate into the three germ layer cell types, thus confirming their ability to generate clinical grade cells. The findings indicated that prolonged culture of IFPSCs (P>6) led to the loss of the stem cell proliferative marker NS, with an increased population doubling time and progression toward neuronal differentiation, acquiring a neurogenic phenotype. Additionally, IFPSCs demonstrated an inherent ability to secrete neurotrophic factors and express receptors for these factors, which is the cause of neuronal differentiation at later passages. Therefore, these findings validated NS as a prognostic indicator for impaired stemness and identified IFPSCs as a promising source for cell-based therapy, particularly for neurodegenerative diseases.

Infrapatellar Fat Pad-Derived Stem Cell-Based Regenerative Strategies in Orthopedic Surgery.

Infrapatellar fat pad is a densely vascularized and innervated extrasynovial tissue that fills the anterior knee compartment. It plays a role in knee biomechanics as well as constitutes a source of stem cells for regeneration after knee injury. Infrapatellar fat pad-derived stem cells (IPFP-ASCs) possess enhanced and age-independent differentiation capacity as compared to other stem cells, which makes them a very promising candidate in stem cell-based regenerative therapy. The aims of this review are to outline the latest advances and potential trends in using IPFP-ASCs and to emphasize the advantages over other sources of stem cells for applications in orthopedic surgery.

Comparative advantages of infrapatellar fat pad: an emerging stem cell source for regenerative medicine.

Growing evidence indicates that infrapatellar fat pad (IPFP)-derived stem cells (IPFSCs) exert robust proliferation capacities and multilineage differentiation potentials. However, few papers summarize the advantages that the IPFP and IPFSCs have in regenerative medicine. In this review we delineate the development and anatomy of the IPFP by comparing it with an adjacent fibrous tissue, synovium, and a more frequently harvested fat depot, subcutaneous adipose tissue. Furthermore, we explore the similarities and differences of stem cells from these three tissues in terms of IPFSCs, synovium-derived stem cells and subcutaneous adipose tissue-derived stem cells in proliferation capacity and tri-lineage differentiation potentials, including chondrogenesis, osteogenesis and adipogenesis. Finally, we highlight the advantages of IPFSCs in regenerative medicine, such as the abundant accessibility and the ability to resist inflammation and senescence, two hurdles for cell-based tissue regeneration. Considering the comparative advantages of IPFSCs, the IPFP can serve as an excellent stem cell source for regenerative medicine, particularly for cartilage regeneration.

Low-oxygen conditions promote synergistic increases in chondrogenesis during co-culture of human osteoarthritic stem cells and chondrocytes.

There has been increased interest in co-cultures of stem cells and chondrocytes for cartilage tissue engineering as there are the limitations associated with using either cell type alone. Drawbacks associated with the use of chondrocytes include the limited numbers of cells available for isolation from damaged or diseased joints, their dedifferentiation during in vitro expansion, and a diminished capacity to synthesise cartilage-specific extracellular matrix components with age and disease. This has motivated the use of adult stem cells with either freshly isolated or culture-expanded chondrocytes for cartilage repair applications; however, the ideal combination of cells and environmental conditions for promoting robust chondrogenesis remains unclear. In this study, we compared the effect of combining a small number of freshly isolated or culture-expanded human chondrocytes with infrapatellar fat pad-derived stem cells (FPSCs) from osteoarthritic donors on chondrogenesis in altered oxygen (5% or 20%) and growth factor supplementation (TGF-β3 only or TGF-β3 and BMP-7) conditions. Both co-cultures, but particularly those including freshly isolated chondrocytes, were found to promote cell proliferation and enhanced matrix accumulation compared to the use of FPSCs alone, resulting in the development of a tissue that was compositionally more similar to that of the native articular cartilage. Local oxygen levels were found to impact chondrogenesis in co-cultures, with more robust increases in proteoglycan and collagen deposition observed at 5% O2 . Additionally, collagen type I synthesis was suppressed in co-cultures maintained at low-oxygen conditions. This study demonstrates that a co-culture of freshly isolated human chondrocytes and FPSCs promotes robust chondrogenesis and thus is a promising cell combination for cartilage tissue engineering.

Meniscus ECM-functionalised hydrogels containing infrapatellar fat pad-derived stem cells for bioprinting of regionally defined meniscal tissue.

Injuries to the meniscus of the knee commonly lead to osteoarthritis. Current therapies for meniscus regeneration, including meniscectomies and scaffold implantation, fail to achieve complete functional regeneration of the tissue. This has led to increased interest in cell and gene therapies and tissue engineering approaches to meniscus regeneration. The implantation of a biomimetic implant, incorporating cells, growth factors, and extracellular matrix (ECM)-derived proteins, represents a promising approach to functional meniscus regeneration. The objective of this study was to develop a range of ECM-functionalised bioinks suitable for 3D bioprinting of meniscal tissue. To this end, alginate hydrogels were functionalised with ECM derived from the inner and outer regions of the meniscus and loaded with infrapatellar fat pad-derived stem cells. In the absence of exogenously supplied growth factors, inner meniscus ECM promoted chondrogenesis of fat pad-derived stem cells, whereas outer meniscus ECM promoted a more elongated cell morphology and the development of a more fibroblastic phenotype. With exogenous growth factors supplementation, a more fibrogenic phenotype was observed in outer ECM-functionalised hydrogels supplemented with connective tissue growth factor, whereas inner ECM-functionalised hydrogels supplemented with TGFβ3 supported the highest levels of Sox-9 and type II collagen gene expression and sulfated glycosaminoglycans (sGAG) deposition. The final phase of the study demonstrated the printability of these ECM-functionalised hydrogels, demonstrating that their codeposition with polycaprolactone microfibres dramatically improved the mechanical properties of the 3D bioprinted constructs with no noticeable loss in cell viability. These bioprinted constructs represent an exciting new approach to tissue engineering of functional meniscal grafts.

Stem cells display a donor dependent response to escalating levels of growth factor release from extracellular matrix-derived scaffolds

Numerous growth factor delivery systems have been developed for tissue engineering. However, little is known about how the dose of a specific protein will influence tissue regeneration, or how different patients will respond to altered levels of growth factor presentation. The objective of the present study was to assess stem cell chondrogenesis within extracellular-matrix (ECM)-derived scaffolds loaded with escalating levels of transforming growth factor (TGF)-β3. It was also sought to determine if stem cells display a donor-dependent response to different doses of TGF-β3, from low (5ng) to high (200ng), released from such scaffolds. It was found that ECM-derived scaffolds possess the capacity to bind and release increasing amounts of TGF-β3, with between 60% and 75% of this growth factor released into the media over the first 12days of culture. After seeding these scaffolds with human infrapatellar fat pad-derived stem cells (FPSCs), it was found that cartilage-specific ECM accumulation was greatest for the higher levels of growth factor loading. Importantly, soak-loading cartilage ECM-derived scaffolds with high levels of TGF-β3 always resulted in at least comparable levels of chondrogenesis to controls where this growth factor was continuously added to the culture media. Similar results were observed for FPSCs from all donors, although the absolute level of secreted matrix did vary from donor to donor. Therefore, while no single growth factor release profile will be optimal for all patients, the results of this study suggest that the combination of a highly porous cartilage ECM-derived scaffold coupled with appropriate levels of TGF-β3 can consistently drive chondrogenesis of adult stem cells. Copyright © 2016 John Wiley & Sons, Ltd.

Study of chondrogenic potential of stem cells in co-culture with chondrons.

Three-dimensional biomimetic scaffolds have widespread applications in biomedical tissue engineering due to similarity of their nanofibrous architecture to native extracellular matrix. Co-culture system has stimulatory effect on chondrogenesis of adult mesenchymal stem cells. This work presents a co-culture strategy using human articular chondrons and adipose-derived stem cells (ASCs) from infrapatellar fat pad (IPFP) for cartilage tissue production. Isolated stem cells were characterized by flowcytometry. Electrospun and polycaprolactone (PCL) scaffolds (900 nm fiber diameter) was obtained from Bon Yakhteh (Tehran-Iran) and human infrapatellar fat pad-derived stem cells (IPFP-ASCs) were seeded on them. IPFP-ASCs on scaffolds were co-cultured with articular chondrons using transwell. After 21 day, chondrogenic differentiation of stem cell was evaluated by determining the genes expression of collagen2, aggrecan and Indian hedgehog using real-time RT-PCR. Genes expression of collagen2, aggrecan by IPFP-ASCs did not alter significantly in comparison with control group. Howevers, expression of Indian hedgehog decreased significantly compared to control group (P< 0.05). These findings indicate that chondrons obtained from osteoarthritic articular cartilage did not stimulate chondrogenic differentiation of IPFP-ASCs in co-culture.

Engineering zonal cartilaginous tissue by modulating oxygen levels and mechanical cues through the depth of infrapatellar fat pad stem cell laden hydrogels.

Engineering tissues with a structure and spatial composition mimicking those of native articular cartilage (AC) remains a challenge. This study examined if infrapatellar fat pad-derived stem cells (FPSCs) can be used to engineer cartilage grafts with a bulk composition and a spatial distribution of matrix similar to the native tissue. In an attempt to mimic the oxygen gradients and mechanical environment within AC, FPSC-laden hydrogels (either 2mm or 4mm in height) were confined to half of their thickness and/or subjected to dynamic compression (DC). Confining FPSC-laden hydrogels was predicted to accentuate the gradient in oxygen tension through the depth of the constructs (higher in the top and lower in the bottom), leading to enhanced glycosaminoglycan (GAG) and collagen synthesis in 2mm high tissues. When subjected to DC alone, both GAG and collagen accumulation increased within 2mm high unconfined constructs. Furthermore, the dynamic modulus of constructs increased from 0.96MPa to 1.45MPa following the application of DC. There was no synergistic benefit of coupling confinement and DC on overall levels of matrix accumulation; however in all constructs, irrespective of their height, the combination of these boundary conditions led to the development of engineered tissues that spatially best resembled native AC. The superficial region of these constructs mimicked that of native tissue, staining weakly for GAG, strongly for type II collagen, and in 4mm high tissues more intensely for proteoglycan 4 (lubricin). This study demonstrated that FPSCs respond to joint-like environmental conditions by producing cartilage tissues mimicking native AC. Copyright © 2016 John Wiley & Sons, Ltd.

Fibrin hydrogels functionalized with cartilage extracellular matrix and incorporating freshly isolated stromal cells as an injectable for cartilage regeneration

Freshly isolated stromal cells can potentially be used as an alternative to in vitro expanded cells in regenerative medicine. Their use requires the development of bioactive hydrogels or scaffolds which provide an environment to enhance their proliferation and tissue-specific differentiation in vivo. The goal of the current study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM microparticles and transforming growth factor (TGF)-β3 as a putative therapeutic for articular cartilage regeneration. ECM microparticles were produced by cryomilling and freeze-drying porcine articular cartilage. Up to 2% (w/v) ECM could be incorporated into fibrin without detrimentally affecting its capacity to form stable hydrogels. To access the chondroinductivity of cartilage ECM, we compared chondrogenesis of infrapatellar fat pad-derived stem cells in fibrin hydrogels functionalized with either particulated ECM or control gelatin microspheres. Cartilage ECM particles could be used to control the delivery of TGF-β3 to IFP-derived stem cells within fibrin hydrogels in vitro, and furthermore, led to higher levels of sulphated glycosaminoglycan (sGAG) and collagen accumulation compared to control constructs loaded with gelatin microspheres. In vivo, freshly isolated stromal cells generated a more cartilage-like tissue within fibrin hydrogels functionalized with cartilage ECM particles compared to the control gelatin loaded constructs. These tissues stained strongly for type II collagen and contained higher levels of sGAGs. These results support the use of fibrin hydrogels functionalized with cartilage ECM components in single-stage, cell-based therapies for joint regeneration. Statement of SignificanceAn alternative to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold or hydrogel is used to provide an environment that enhances their proliferation and tissue-specific differentiation in vivo. The objective of this study was to develop an injectable fibrin hydrogel functionalized with cartilage ECM micro-particles and the growth factor TGF-β3 as a therapeutic for articular cartilage regeneration. This study demonstrates that freshly isolated stromal cells generate cartilage tissue in vivo when incorporated into such a fibrin hydrogels functionalized with cartilage ECM particles. These findings open up new possibilities for in-theatre, single-stage, cell-based therapies for joint regeneration.

Intermittent hydrostatic pressure maintains and enhances the chondrogenic differentiation of cartilage progenitor cells cultivated in alginate beads.

The objective of this study was to explore the effects of intermittent hydrostatic pressure (IHP) on the chondrogenic differentiation of cartilage progenitor cells (CPCs) cultivated in alginate beads. CPCs were isolated from the knee joint cartilage of rabbits, and infrapatellar fat pad-derived stem cells (FPSCs) and chondrocytes (CCs) were included as the control cell types. Cells embedded in alginate beads were treated with IHP at 5Mpa and 0.5Hz for 4h/day for 1, 2, or 4weeks. The cells' migratory and proliferative capacities were evaluated using the scratch and Live/Dead assays, respectively. Hematoxylin and eosin staining, safranin O staining, and immunohistochemical staining were performed to determine the effects of IHP on the synthesis of extracellular matrix (ECM) proteins. Real-time polymerase chain reaction analysis was performed to measure the expression of genes related to chondrogenesis. The scratch and Live/Dead assays revealed that IHP significantly promoted the migration and proliferation of FPSCs and CPCs to different extents. The staining experiments showed greater production of cartilage ECM components (glycosaminoglycans and collagen II) by cells exposed to IHP, and the gene expression analysis demonstrated that IHP stimulated the expression of chondrocyte-related genes. Importantly, these effects of IHP were more prominent in CPCs than in FPSCs and CCs. Considering all of our experimental results combined, we conclude that CPCs demonstrated a stronger chondrogenic differentiation capacity than the FPSCs and CCs under stimulation with IHP. Thus, the use of CPCs, combined with mechanical stimulation, may represent a valuable strategy for cartilage tissue engineering.

Engineering cartilaginous grafts using chondrocyte-laden hydrogels supported by a superficial layer of stem cells.

During postnatal joint development, progenitor cells that reside in the superficial region of articular cartilage first drive the rapid growth of the tissue and later help direct the formation of mature hyaline cartilage. These developmental processes may provide directions for the optimal structuring of co-cultured chondrocytes (CCs) and multipotent stromal/stem cells (MSCs) required for engineering cartilaginous tissues. The objective of this study was to engineer cartilage grafts by recapitulating aspects of joint development where a population of superficial progenitor cells drives the development of the tissue. To this end, MSCs were either self-assembled on top of CC-laden agarose gels (structured co-culture) or were mixed with CCs before being embedded in an agarose hydrogel (mixed co-culture). Porcine infrapatellar fat pad-derived stem cells (FPSCs) and bone marrow-derived MSCs (BMSCs) were used as sources of progenitor cells. The DNA, sGAG and collagen content of a mixed co-culture of FPSCs and CCs was found to be lower than the combined content of two control hydrogels seeded with CCs and FPSCs only. In contrast, a mixed co-culture of BMSCs and CCs led to increased proliferation and sGAG and collagen accumulation. Of note was the finding that a structured co-culture, at the appropriate cell density, led to greater sGAG accumulation than a mixed co-culture for both MSC sources. In conclusion, assembling MSCs onto CC-laden hydrogels dramatically enhances the development of the engineered tissue, with the superficial layer of progenitor cells driving CC proliferation and cartilage ECM production, mimicking certain aspects of developing cartilage. Copyright © 2015 John Wiley & Sons, Ltd.

Coupling Freshly Isolated CD44+ Infrapatellar Fat Pad‐Derived Stromal Cells with a TGF‐β3 Eluting Cartilage ECM‐Derived Scaffold as a Single‐Stage Strategy for Promoting Chondrogenesis

An alternative strategy to the use of in vitro expanded cells in regenerative medicine is the use of freshly isolated stromal cells, where a bioactive scaffold is used to provide an environment conducive to proliferation and tissue-specific differentiation in vivo. The objective of this study is to develop a cartilage extracellular matrix (ECM)-derived scaffold that could facilitate the rapid proliferation and chondrogenic differentiation of freshly isolated stromal cells. By freeze-drying cryomilled cartilage ECM of differing concentrations, it is possible to produce scaffolds with a range of pore sizes. The migration, proliferation, and chondrogenic differentiation of infrapatellar fat pad-derived stem cells (FPSCs) depend on the concentration/porosity of these scaffolds, with greater sulphated glycosaminoglycan (sGAG) accumulation observed in scaffolds with larger-sized pores. It is then sought to determine if freshly isolated fat pad-derived stromal cells, seeded onto a transforming growth factor (TGF)-β3 eluting ECM-derived scaffold, could promote chondrogenesis in vivo. While a more cartilage-like tissue could be generated using culture expanded FPSCs compared to nonenriched freshly isolated cells, fresh CD44(+) stromal cells are capable of producing a tissue in vivo that stained strongly for sGAGs and type II collagen. These findings open up new possibilities for in-theatre cell-based therapies for joint regeneration.

Infrapatellar fat pad-derived stem cells maintain their chondrogenic capacity in disease and can be used to engineer cartilaginous grafts of clinically relevant dimensions.

A therapy for regenerating large cartilaginous lesions within the articular surface of osteoarthritic joints remains elusive. While tissue engineering strategies such as matrix-assisted autologous chondrocyte implantation can be used in the repair of focal cartilage defects, extending such approaches to the treatment of osteoarthritis will require a number of scientific and technical challenges to be overcome. These include the identification of an abundant source of chondroprogenitor cells that maintain their chondrogenic capacity in disease, as well as the development of novel approaches to engineer scalable cartilaginous grafts that could be used to resurface large areas of damaged joints. In this study, it is first demonstrated that infrapatellar fat pad-derived stem cells (FPSCs) isolated from osteoarthritic (OA) donors possess a comparable chondrogenic capacity to FPSCs isolated from patients undergoing ligament reconstruction. In a further validation of their functionality, we also demonstrate that FPSCs from OA donors respond to the application of physiological levels of cyclic hydrostatic pressure by increasing aggrecan gene expression and the production of sulfated glycosaminoglycans. We next explored whether cartilaginous grafts could be engineered with diseased human FPSCs using a self-assembly or scaffold-free approach. After examining a range of culture conditions, it was found that continuous supplementation with both transforming growth factor-β3 (TGF-β3) and bone morphogenic protein-6 (BMP-6) promoted the development of tissues rich in proteoglycans and type II collagen. The final phase of the study sought to scale-up this approach to engineer cartilaginous grafts of clinically relevant dimensions (≥2 cm in diameter) by assembling FPSCs onto electrospun PLLA fiber membranes. Over 6 weeks in culture, it was possible to generate robust, flexible cartilage-like grafts of scale, opening up the possibility that tissues engineered using FPSCs derived from OA patients could potentially be used to resurface large areas of joint surfaces damaged by trauma or disease.

Engineering articular cartilage‐like grafts by self‐assembly of infrapatellar fat pad‐derived stem cells

Well documented limitations associated with primary chondrocytes for cartilage tissue engineering applications have led to increased interest in the use of multi-potent stem/progenitor cells. The objective of this study was to firstly investigate if infrapatellar fat pad-derived stem cells (FPSCs) could be used to engineer cartilage-like tissues through a self-assembly (SA) process, and secondly to compare the properties of such grafts to those engineered by agarose hydrogel encapsulation (AE). Self-assembled cartilaginous tissues were first engineered by geometrically confining FPSCs on tissue culture plastic, and then either continuously or transiently supplementing these constructs with transforming growth factor-b3 (TGF-b3). Transient supplementation with TGF-b3 (for the first 21 days of culture) enhanced the development of self-assembled grafts, with sGAG accumulation reaching levels of 8.4 ± 1.5% w/w after 6 weeks of culture. While overall levels of matrix synthesis were higher with AE compared to SA, when normalized to tissue wet weight, ECM accumulation was significantly greater in the lighter SA constructs. A potential drawback with the SA approach on tissue culture plastic was that it often led to the development of contracted,geometrically inconsistent tissues.We therefore next explored if SA on polyethylene terephthalate (PET) transwell membranes would lead to the development of more morphologically stable and homogenous tissues. At high seeding densities, SA on such transwell membranes led to the formation of geometrically uniform constructs that underwent minimal contraction during culture. In conclusion, the results of this study demonstrate the potential of SA using FPSCs for cartilage tissue engineering, with grafts attaining relatively high levels of sGAG content within clinically relevant timeframes. Such an approach is easily scalable and may lend itself to treating large, full thickness cartilage defects.