Sustained delivery of bioactive TGF-β1 from self-assembling peptide hydrogels induces chondrogenesis of encapsulated bone marrow stromal cells

Tissue engineering strategies for cartilage defect repair require technology for local targeted delivery of chondrogenic and anti-inflammatory factors. The objective of this study was to determine the release kinetics of transforming growth factor β1 (TGF-β1) from self-assembling peptide hydrogels, a candidate scaffold for cell transplant therapies, and stimulate chondrogenesis of encapsulated young equine bone marrow stromal cells (BMSCs). Although both peptide and agarose hydrogels retained TGF-β1, fivefold higher retention was found in peptide. Excess unlabeled TGF-β1 minimally displaced retained radiolabeled TGF-β1, demonstrating biologically relevant loading capacity for peptide hydrogels. The initial release from acellular peptide hydrogels was nearly threefold lower than agarose hydrogels, at 18% of loaded TGF-β1 through 3 days as compared to 48% for agarose. At day 21, cumulative release of TGF-β1 was 32–44% from acellular peptide hydrogels, but was 62% from peptide hydrogels with encapsulated BMSCs, likely due to cell-mediated TGF-β1 degradation and release of small labeled species. TGF-β1 loaded peptide hydrogels stimulated chondrogenesis of young equine BMSCs, a relevant preclinical model for treating injuries in young human cohorts. Self-assembling peptide hydrogels can be used to deliver chondrogenic factors to encapsulated cells making them a promising technology for in vivo, cell-based regenerative medicine.National Institutes of Health (U.S.) (NIH EB003805)National Institutes of Health (U.S.) (NIH AR60331)National Institutes of Health (U.S.). Molecular, Cell, and Tissue Biomechanics (Training Grant Fellowship)Arthritis Foundation (postdoctoral fellowship

Effect of self-assembling peptide, chondrogenic factors, and bone marrow-derived stromal cells on osteochondral repair

Objective The goal of this study was to test the ability of an injectable self-assembling peptide (KLD) hydrogel with or without chondrogenic factors (CF) and allogeneic bone marrow stromal cells (BMSCs) to stimulate cartilage regeneration in a full-thickness, critically-sized, rabbit cartilage defect model in vivo. We used CF treatments to test the hypotheses that CF would stimulate chondrogenesis and matrix production by cells migrating into acellular KLD (KLD + CF) or by BMSCs delivered in KLD (KLD + CF + BMSCs). Design Three groups were tested against contralateral untreated controls: KLD, KLD + CF, and KLD + CF +BMSCs, n = 6–7. Transforming growth factor-β1 (TGF-β1), dexamethasone, and insulin-like growth factor-1 (IGF-1) were used as CF pre-mixed with KLD and BMSCs before injection. Evaluations included gross, histological, immunohistochemical and radiographic analyses. Results KLD without CF or BMSCs showed the greatest repair after 12 weeks with significantly higher Safranin-O, collagen II immunostaining, and cumulative histology scores than untreated contralateral controls. KLD + CF resulted in significantly higher aggrecan immunostaining than untreated contralateral controls. Including allogeneic BMSCs + CF markedly reduced the quality of repair and increased osteophyte formation compared to KLD-alone. Conclusions These data show that KLD can fill full-thickness osteochondral defects in situ and improve cartilage repair as shown by Safranin-O, collagen II immunostaining, and cumulative histology. In this small animal model, the full-thickness critically-sized defect provided access to the marrow, similar in concept to abrasion arthroplasty or spongialization in large animal models, and suggests that combining KLD with these techniques may improve current practice.National Institutes of Health (U.S.) (National Institute for Biomedical Imaging and Bioengineering (U.S.) Grant EB003805)American Society for Engineering Education. National Defense Science and Engineering Graduate FellowshipNational Science Foundation (U.S.). Graduate Research FellowshipNational Institutes of Health (U.S.) (Grant EB003805)National Institutes of Health (U.S.) (Grant AR33236)Arthritis Foundation (Postdoctoral Fellowship

Treatment Effects of Intra-Articular Allogenic Mesenchymal Stem Cell Secretome in an Equine Model of Joint Inflammation

Background: Allogenic mesenchymal stem cell (MSC) secretome is a novel intra-articular therapeutic that has shown promise in in vitro and small animal models and warrants further investigation. Objectives: To investigate if intra-articular allogenic MSC-secretome has anti-inflammatory effects using an equine model of joint inflammation. Study Design: Randomized positively and negatively controlled experimental study. Method: In phase 1, joint inflammation was induced bilaterally in radiocarpal joints of eight horses by injecting 0.25 ng lipopolysaccharide (LPS). After 2 h, the secretome of INFy and TNFα stimulated allogeneic equine MSCs was injected in one randomly assigned joint, while the contralateral joint was injected with medium (negative control). Clinical parameters (composite welfare scores, joint effusion, joint circumference) were recorded, and synovial fluid samples were analyzed for biomarkers (total protein, WBCC; eicosanoid mediators, CCL2; TNFα; MMP; GAGs; C2C; CPII) at fixed post-injection hours (PIH 0, 8, 24, 72, and 168 h). The effects of time and treatment on clinical and synovial fluid parameters and the presence of time-treatment interactions were evaluated. For phase 2, allogeneic MSC-secretome vs. allogeneic equine MSCs (positive control) was tested using a similar methodology. Results: In phase 1, the joint circumference was significantly (p < 0.05) lower in the MSC-secretome treated group compared to the medium control group at PIH 24, and significantly higher peak synovial GAG values were noted at PIH 24 (p < 0.001). In phase 2, no significant differences were noted between the treatment effects of MSC-secretome and MSCs. Main Limitations: This study is a controlled experimental study and therefore cannot fully reflect natural joint disease. In phase 2, two therapeutics are directly compared and there is no negative control. Conclusions: In this model of joint inflammation, intra-articular MSC-secretome injection had some clinical anti-inflammatory effects. An effect on cartilage metabolism, evident as a rise in GAG levels was also noted, although it is unclear whether this could be considered a beneficial or detrimental effect. When directly comparing MSC-secretome to MSCs in this model results were comparable, indicating that MSC-secretome could be a viable off-the-shelf alternative to MSC treatment

In vitro culture of a chondrocyte-seeded peptide hydrogel and the effects of dynamic compression

Thesis (Ph. D. in Bioengineering)--Massachusetts Institute of Technology, Biological Engineering Division, 2003.Includes bibliographical references.Emerging medical technologies for effective and lasting repair of articular cartilage include delivery of cells or cell-seeded scaffolds to a defect site to initiate de novo tissue regeneration. Biocompatible scaffolds assist in providing a template for cell distribution and extracellular matrix accumulation in a three-dimensional geometry. In these studies, a self-assembling peptide hydrogel is evaluated as a potential scaffold for cartilage repair using a model bovine cell source. A seeding technique is developed for 3-D encapsulation of chondrocytes in a peptide hydrogel. The chondrocyte-seeded peptide hydrogel was then evaluated cellular activities in vitro under standard culture conditions and also when subjected to dynamic compression. During 4 weeks of culture in vitro, chondrocytes seeded within the peptide hydrogel retained their morphology and developed a cartilage-like ECM rich in proteoglycans and type II collagen, indicative of a stable chondrocyte phenotype. Time dependent accumulation of this ECM was paralleled by increases in material stiffness, indicative of deposition of mechanically-functional neo-tissue. Culture of chondrocyte-seeded peptide hydrogels in ITS-supplemented medium was investigated as an alternative to high serum culture. Low serum (0.2%), ITS-supplemented medium was found to maintain high levels of cell division and extracellular matrix synthesis and accumulation, as seen in high serum culture. Furthermore, low serum, ITS medium induced minimal chondrocyte de-differentiation on the surface of the hydrogel. This is in contrast to high serum culture, where surface de-differentiation and subsequent proliferation led to a 5-10 cell thick layer that stained positive for type I collagen.(cont.) The effects of dynamic compression of chondrocyte-seeded peptide hydrogels were evaluated over long-term culture. A non-continuous loading protocol was identified in which proteoglycan, but not protein, synthesis increased over static, free-swelling culture. Increases in GAG matrix accumulation were observed after at least 8 days of loading, while hydroxyproline accumulation was unaffected by dynamic compression. These data demonstrated dynamic compression differentially regulated the synthesis of proteoglycans. Analysis of GAG loss to the medium indicated peak proteoglycan catabolism occurred immediately after the initiation of loading. This phenomenon was further explored using a modified loading protocol that increased GAG loss to the medium. Peak GAG loss to the medium was 2-fold higher than previously observed, resulting in GAG accumulation values significantly less than controls. Hydroxyproline accumulation was minimally affected by loading, demonstrating that dynamic compression also differentially regulated the catabolism of proteoglycans. Proteoglycan catabolism was not predominantly due to physical disruption accumulated extracellular matrix or loss of newly-synthesized molecules. Instead, the presence of MMPs in the medium that coincided with GAG loss suggest a potential enzymatic mechanism. These results demonstrate the potential of a self-assembling peptide hydrogel as a scaffold for the synthesis and accumulation of a true cartilage-like extracellular matrix ...John D. Kisiday.Ph.D.in Bioengineerin

Dynamic Compression Stimulates Proteoglycan Synthesis by Mesenchymal Stem Cells in the Absence of Chondrogenic Cytokines

The objective of this study was to evaluate the effect of dynamic compression on mesenchymal stem cell (MSC) chondrogenesis. Dynamic compression was applied to agarose hydrogels seeded with bone marrow-derived adult equine MSCs. In the absence of the chondrogenic cytokine transforming growth factor beta (TGFb), dynamic compression applied for 12 h per day led to significantly greater proteoglycan synthesis than in unloaded TGFb-free cultures, although at a rate that was approximately 20% to 35% of unloaded TGFb cultures. These data suggest that the emergence of aggrecan dominated a chondrogenic response to loading as increases in proteoglycan synthesis. Cross-sectional analyses were conducted to subjectively identify potential spatial distributions of heterogeneous differentiation. In loaded samples, cell viability and metachromatic staining was low near the porous compression platen interface but increased with depth, reaching levels in the lower portion of the hydrogel that resembled unloaded TGFb cultures. These results suggest that the combination of high hydrostatic pressure and low dynamic strain and fluid flow had a stronger effect on chondrogenesis than did low hydrostatic pressure coupled with high dynamic strain and fluid flow. Next, the 12-h per day loading protocol was applied in the presence of TGFb. Biosynthesis in loaded cultures was less than in unloaded TGFb samples. Taken together, these data suggest that the duration of loading necessary to stimulate mechanoinduction of MSCs may not be optimal for neo-tissue accumulation in the presence of chondrogenic cytokines.National Institutes of Health (U.S.). Bioengineering Research Partnership (Grant EB003805)National Institutes of Health (U.S.) (NIH grant AR33236

Controlled Delivery of Transforming Growth Factor β1 [beta 1] by Self-Assembling Peptide Hydrogels Induces Chondrogenesis of Bone Marrow Stromal Cells and Modulates Smad2/3 Signaling

Self-assembling peptide hydrogels were modified to deliver transforming growth factor b1 [beta 1](TGF-b1)[TGF beta 1] to encapsulated bone-marrow-derived stromal cells (BMSCs) for cartilage tissue engineering applications using two different approaches: (i) biotin-streptavidin tethering; (ii) adsorption to the peptide scaffold. Initial studies to determine the duration of TGF-b1 [TGF beta 1] medium supplementation necessary to stimulate chondrogenesis showed that 4 days of transient soluble TGF-b1 [TGF beta 1] to newborn bovine BMSCs resulted in 10-fold higher proteoglycan accumulation than TGF-b1-free [TGF beta 1 free]culture after 3 weeks. Subsequently, BMSC-seeded peptide hydrogels with either tethered TGF-b1 [TGF beta 1] (Teth-TGF) or adsorbed TGF-b1 [TGF beta 1] (Ads-TGF) were cultured in the TGF-b1-free [TGF beta 1 free] medium, and chondrogenesis was compared to that for BMSCs encapsulated in unmodified peptide hydrogels, both with and without soluble TGF-b1 [TGF beta 1] medium supplementation. Ads-TGF peptide hydrogels stimulated chondrogenesis of BMSCs as demonstrated by cell proliferation and cartilage-like extracellular matrix accumulation, whereas Teth- TGF did not stimulate chondrogenesis. In parallel experiments, TGF-b1 [TGF beta 1] adsorbed to agarose hydrogels stimulated comparable chondrogenesis. Full-length aggrecan was produced by BMSCs in response to Ads-TGF in both peptide and agarose hydrogels, whereas medium-delivered TGF-b1 [TGF beta 1] stimulated catabolic aggrecan cleavage product formation in agarose but not peptide scaffolds. Smad2/3 was transiently phosphorylated in response to Ads-TGF but not Teth-TGF, whereas medium-delivered TGF-b1 [TGF beta 1] produced sustained signaling, suggesting that dose and signal duration are potentially important for minimizing aggrecan cleavage product formation. Robustness of this technology for use in multiple species and ages was demonstrated by effective chondrogenic stimulation of adult equine BMSCs, an important translational model used before the initiation of human clinical studies.National Institutes of Health (U.S.) ( (NIH EB003805) (NIH AR33236) (NIH AR45779)National Institutes of Health (U.S.). Molecular, Cell, and Tissue Biomechanics Training Grant FellowshipArthritis Foundatio

Adult equine bone-marrow stromal cells produce a cartilage-like ECM superior to animal-matched adult chondrocytes

Our objective was to evaluate the age-dependent mechanical phenotype of bone marrow stromal cell- (BMSC-) and chondrocyte-produced cartilage-like neo-tissue and to elucidate the matrix-associated mechanisms which generate this phenotype. Cells from both immature (2–4 month-old foals) and skeletally-mature (2–5 year-old adults) mixed-breed horses were isolated from animal-matched bone marrow and cartilage tissue, encapsulated in self-assembling-peptide hydrogels, and cultured with and without TGF-β1 supplementation. BMSCs and chondrocytes from both donor ages were encapsulated with high viability. BMSCs from both ages produced neo-tissue with higher mechanical stiffness than that produced by either young or adult chondrocytes. Young, but not adult, chondrocytes proliferated in response to TGF-β1 while BMSCs from both age groups proliferated with TGF-β1. Young chondrocytes stimulated by TGF-β1 accumulated ECM with 10-fold higher sulfated-glycosaminoglycan content than adult chondrocytes and 2–3-fold higher than BMSCs of either age. The opposite trend was observed for hydroxyproline content, with BMSCs accumulating 2–3-fold more than chondrocytes, independent of age. Size-exclusion chromatography of extracted proteoglycans showed that an aggrecan-like peak was the predominant sulfated proteoglycan for all cell types. Direct measurement of aggrecan core protein length and chondroitin sulfate chain length by single molecule atomic force microscopy imaging revealed that, independent of age, BMSCs produced longer core protein and longer chondroitin sulfate chains, and fewer short core protein molecules than chondrocytes, suggesting that the BMSC-produced aggrecan has a phenotype more characteristic of young tissue than chondrocyte-produced aggrecan. Aggrecan ultrastructure, ECM composition, and cellular proliferation combine to suggest a mechanism by which BMSCs produce a superior cartilage-like neo-tissue than either young or adult chondrocytes.National Institutes of Health (U.S.) (EB003805)National Institutes of Health (U.S.) (AR33236)National Science Foundation (U.S.) (NSF-NIRT 0403903)National Institutes of Health (U.S.). Molecular, Cell, and Tissue Biomechanics Training Grant FellowshipArthritis Foundatio

Growth Factor-Mediated Migration of Bone Marrow Progenitor Cells for Accelerated Scaffold Recruitment

Tissue engineering approaches using growth factor-functionalized acellular scaffolds to support and guide repair driven by endogenous cells are thought to require a careful balance between cell recruitment and growth factor release kinetics. The objective of this study was to identify a growth factor combination that accelerates progenitor cell migration into self-assembling peptide hydrogels in the context of cartilage defect repair. A novel 3D gel-to-gel migration assay enabled quantification of the chemotactic impact of platelet-derived growth factor-BB (PDGF-BB), heparin-binding insulin-like growth factor-1 (HB-IGF-1), and transforming growth factor-β1 (TGF-β1) on progenitor cells derived from subchondral bovine trabecular bone (bone-marrow progenitor cells, BM-PCs) encapsulated in the peptide hydrogel [KLDL]3. Only the combination of PDGF-BB and TGF-β1 stimulated significant migration of BM-PCs over a 4-day period, measured by confocal microscopy. Both PDGF-BB and TGF-β1 were slowly released from the gel, as measured using their 125I-labeled forms, and they remained significantly present in the gel at 4 days. In the context of augmenting microfracture surgery for cartilage repair, our strategy of delivering chemotactic and proanabolic growth factors in KLD may provide the necessary local stimulus to help increase defect cellularity, providing more cells to generate repair tissue.National Institutes of Health (U.S.) (Grant AR060331

Growth Factor-Mediated Migration of Bone Marrow Progenitor Cells for Accelerated Scaffold Recruitment

Tissue engineering approaches using growth factor-functionalized acellular scaffolds to support and guide repair driven by endogenous cells are thought to require a careful balance between cell recruitment and growth factor release kinetics. The objective of this study was to identify a growth factor combination that accelerates progenitor cell migration into self-assembling peptide hydrogels in the context of cartilage defect repair. A novel 3D gel-to-gel migration assay enabled quantification of the chemotactic impact of platelet-derived growth factor-BB (PDGF-BB), heparin-binding insulin-like growth factor-1 (HB-IGF-1), and transforming growth factor-β1 (TGF-β1) on progenitor cells derived from subchondral bovine trabecular bone (bone-marrow progenitor cells, BM-PCs) encapsulated in the peptide hydrogel [KLDL]3. Only the combination of PDGF-BB and TGF-β1 stimulated significant migration of BM-PCs over a 4-day period, measured by confocal microscopy. Both PDGF-BB and TGF-β1 were slowly released from the gel, as measured using their 125I-labeled forms, and they remained significantly present in the gel at 4 days. In the context of augmenting microfracture surgery for cartilage repair, our strategy of delivering chemotactic and proanabolic growth factors in KLD may provide the necessary local stimulus to help increase defect cellularity, providing more cells to generate repair tissue.National Institutes of Health (U.S.) (Grant AR060331