Cytex Publications Overview 2023

1. Moutos FT, Freed LE, Guilak F. A biomimetic three-dimensional woven composite scaffold for functional tissue engineering of cartilage. Nat Mater. 2007;6(2):162-7. Epub 2007/01/24. doi: 10.1038/nmat1822. PubMed PMID: 17237789. https://pubmed.ncbi.nlm.nih.gov/17237789/

Abstract: Tissue engineering seeks to repair or regenerate tissues through combinations of implanted cells, biomaterial scaffolds and biologically active molecules. The rapid restoration of tissue biomechanical function remains an important challenge, emphasizing the need to replicate structural and mechanical properties using novel scaffold designs. Here we present a microscale 3D weaving technique to generate anisotropic 3D woven structures as the basis for novel composite scaffolds that are consolidated with a chondrocyte-hydrogel mixture into cartilage tissue constructs. Composite scaffolds show mechanical properties of the same order of magnitude as values for native articular cartilage, as measured by compressive, tensile and shear testing. Moreover, our findings showed that porous composite scaffolds could be engineered with initial properties that reproduce the anisotropy, viscoelasticity and tension-compression nonlinearity of native articular cartilage. Such scaffolds uniquely combine the potential for load-bearing immediately after implantation in vivo with biological support for cell-based tissue regeneration without requiring cultivation in vitro.

2. Moutos FT, Guilak F. Composite scaffolds for cartilage tissue engineering. Biorheology. 2008;45(3- 4):501-12. Epub 2008/10/07. PubMed PMID: 18836249; PMCID: PMC2727640.

Abstract: Tissue engineering remains a promising therapeutic strategy for the repair or regeneration of diseased or damaged tissues. Previous approaches have typically focused on combining cells and bioactive molecules (e.g., growth factors, cytokines and DNA fragments) with a biomaterial scaffold that functions as a template to control the geometry of the newly formed tissue, while facilitating the attachment, proliferation, and differentiation of embedded cells. Biomaterial scaffolds also play a crucial role in determining the functional properties of engineered tissues, including biomechanical characteristics such as inhomogeneity, anisotropy, nonlinearity or viscoelasticity. While single-phase, homogeneous materials have been used extensively to create numerous types of tissue constructs, there continue to be significant challenges in the development of scaffolds that can provide the functional properties of load-bearing tissues such as articular cartilage. In an attempt to create more complex scaffolds that promote the regeneration of functional engineered tissues, composite scaffolds comprising two or more distinct materials have been developed. This paper reviews various studies on the development and testing of composite scaffolds for the tissue engineering of articular cartilage, using techniques such as embedded fibers and textiles for reinforcement, embedded solid structures, multi- layered designs, or three-dimensionally woven composite materials. In many cases, the use of composite scaffolds can provide unique biomechanical and biological properties for the development of functional tissue engineering scaffolds.

3. Freed LE, Engelmayr GC, Jr., Borenstein JT, Moutos FT, Guilak F. Advanced material strategies for tissue engineering scaffolds. Adv Mater. 2009;21(32-33):3410-8. Epub 2010/10/01. doi: 10.1002/adma.200900303. PubMed PMID: 20882506; PMCID: PMC3003664

Abstract: Tissue engineering seeks to restore the function of diseased or damaged tissues through the use of cells and biomaterial scaffolds. It is now apparent that the next generation of functional tissue replacements will require advanced material strategies to achieve many of the important requirements for long-term success. Here we provide representative examples of engineered skeletal and myocardial tissue constructs in which scaffolds were explicitly designed to match native tissue mechanical properties as well as to promote cell alignment. We discuss recent progress in microfluidic devices that can potentially serve as tissue engineering scaffolds, since mass transport via microvascular-like structures will be essential in the development of tissue engineered constructs on the length scale of native tissues. Given the rapid evolution of the field of tissue engineering, it is important to consider the use of advanced materials in light of the emerging role of genetics, growth factors, bioreactors, and other technologies.

4. Guilak F, Estes BT, Diekman BO, Moutos FT, Gimble JM. 2010 Nicolas Andry Award: Multipotent adult stem cells from adipose tissue for musculoskeletal tissue engineering. Clin Orthop Relat Res. 2010;468(9):2530-40. Epub 2010/07/14. doi: 10.1007/s11999-010-14109. PubMed PMID: 20625952; PMCID: PMC2919887.

Abstract: 

BACKGROUND: Cell-based therapies such as tissue engineering provide promising therapeutic possibilities to enhance the repair or regeneration of damaged or diseased tissues but are dependent on the availability and controlled manipulation of appropriate cell sources.

QUESTIONS/PURPOSES: The goal of this study was to test the hypothesis that adult subcutaneous fat contains stem cells with multilineage potential and to determine the influence of specific soluble mediators and biomaterial scaffolds on their differentiation into musculoskeletal phenotypes.

METHODS: We reviewed recent studies showing the stem-like characteristics and multipotency of adipose-derived stem cells (ASCs), and their potential application in cell-based therapies in orthopaedics.

RESULTS: Under controlled conditions, ASCs show phenotypic characteristics of various cell types, including chondrocytes, osteoblasts, adipocytes, neuronal cells, or muscle cells. In particular, the chondrogenic differentiation of ASCs can be induced by low oxygen tension, growth factors such as bone morphogenetic protein-6 (BMP-6), or biomaterial scaffolds consisting of native tissue matrices derived from cartilage. Finally, focus is given to the development of a functional biomaterial scaffold that can provide ASC-based constructs with mechanical properties similar to native cartilage.

CONCLUSIONS: Adipose tissue contains an abundant source of multipotent progenitor cells. These cells show cell surface marker profiles and differentiation characteristics that are similar to but distinct from other adult stem cells, such as bone marrow mesenchymal stem cells (MSCs).

CLINICAL RELEVANCE: The availability of an easily accessible and reproducible cell source may greatly facilitate the development of new cell-based therapies for regenerative medicine applications in the musculoskeletal system.

5. Moutos FT, Estes BT, Guilak F. Multifunctional hybrid three-dimensionally woven scaffolds for cartilage tissue engineering. Macromol Biosci. 2010;10(11):1355-64. Epub 2010/09/22. doi: 10.1002/mabi.201000124. PubMed PMID: 20857388; PMCID: PMC3214627.

Abstract: The successful replacement of large-scale cartilage defects or osteoarthritic lesions using tissue- engineering approaches will likely require composite biomaterial scaffolds that have biomimetic mechanical properties and can provide cell-instructive cues to control the growth and differentiation of embedded stem or progenitor cells. This study describes a novel method of constructing multifunctional scaffolds for cartilage tissue engineering that can provide both mechanical support and biological stimulation to seeded progenitor cells. 3-D woven PCL scaffolds were infiltrated with a slurry of homogenized CDM of porcine origin, seeded with human ASCs, and cultured for up to 42 d under standard growth conditions. These constructs were compared to scaffolds derived solely from CDM as well as 3-D woven PCL fabric without CDM. While all scaffolds promoted a chondrogenic phenotype of the ASCs, CDM scaffolds showed low compressive and shear moduli and contracted significantly during culture. Fiber-reinforced CDM scaffolds and 3-D woven PCL scaffolds maintained their mechanical properties throughout the culture period, while supporting the accumulation of a cartilaginous extracellular matrix. These findings show that fiber- reinforced hybrid scaffolds can be produced with biomimetic mechanical properties as well as the ability to promote ASC differentiation and chondrogenesis in vitro.

6. Moutos FT, Guilak F. Functional properties of cell-seeded three-dimensionally woven poly(epsilon- caprolactone) scaffolds for cartilage tissue engineering. Tissue Eng Part A. 2010;16(4):1291-301. Epub 2009/11/12. doi: 10.1089/ten.TEA.2009.0480. PubMed PMID: 19903085; PMCID: PMC2862608.

Abstract: Articular cartilage possesses complex mechanical properties that provide healthy joints the ability to bear repeated loads and maintain smooth articulating surfaces over an entire lifetime. In this study, we utilized a fiber-reinforced composite scaffold designed to mimic the anisotropic, nonlinear, and viscoelastic biomechanical characteristics of native cartilage as the basis for developing functional tissue-engineered constructs. Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were encapsulated with a fibrin hydrogel, seeded with human adipose-derived stem cells, and cultured for 28 days in chondrogenic culture conditions. Biomechanical testing showed that PCL-based constructs exhibited baseline compressive and shear properties similar to those of native cartilage and maintained these properties throughout the culture period, while supporting the synthesis of a collagen-rich extracellular matrix. Further, constructs displayed an equilibrium coefficient of friction similar to that of native articular cartilage (mu(eq) approximately 0.1- 0.3) over the prescribed culture period. Our findings show that three-dimensionally woven PCL-fibrin composite scaffolds can be produced with cartilage-like mechanical properties, and that these engineered properties can be maintained in culture while seeded stem cells regenerate a new, functional tissue construct.

7. Valonen PK, Moutos FT, Kusanagi A, Moretti MG, Diekman BO, Welter JF, Caplan AI, Guilak F, Freed LE. In vitro generation of mechanically functional cartilage grafts based on adult human stem cells and 3D- woven poly(epsilon-caprolactone) scaffolds. Biomaterials. 2010;31(8):2193-200. Epub 2009/12/26. doi: 10.1016/j.biomaterials.2009.11.092. PubMed PMID: 20034665; PMCID: PMC2824534.

Abstract: Three-dimensionally woven poly(epsilon-caprolactone) (PCL) scaffolds were combined with adult human mesenchymal stem cells (hMSC) to engineer mechanically functional cartilage constructs in vitro. The specific objectives were to: (i) produce PCL scaffolds with cartilage- like mechanical properties, (ii) demonstrate that hMSCs formed cartilage after 21 days of culture on PCL scaffolds, and (iii) study effects of scaffold structure (loosely vs. tightly woven), culture vessel (static dish vs. oscillating bioreactor), and medium composition (chondrogenic additives with or without serum). Aggregate moduli of 21-day constructs approached normal articular cartilage for tightly woven PCL cultured in bioreactors, were lower for tightly woven PCL cultured statically, and lowest for loosely woven PCL cultured statically (p<0.05). Construct DNA, total collagen, and glycosaminoglycans (GAG) increased in a manner dependent on time, culture vessel, and medium composition. Chondrogenesis was verified histologically by rounded cells within a hyaline-like matrix that immunostained for collagen type II but not type I. Bioreactors yielded constructs with higher collagen content (p<0.05) and more homogenous matrix than static controls. Chondrogenic additives yielded constructs with higher GAG (p<0.05) and earlier expression of collagen II mRNA if serum was not present in medium. These results show feasibility of functional cartilage tissue engineering from hMSC and 3D-woven PCL scaffolds.

https://pubmed.ncbi.nlm.nih.gov/20034665/

8. Ousema PH, Moutos FT, Estes BT, Caplan AI, Lennon DP, Guilak F, Weinberg JB. The inhibition by interleukin 1 of MSC chondrogenesis and the development of biomechanical properties in biomimetic 3D woven PCL scaffolds. Biomaterials. 2012;33(35):8967-74. Epub 2012/09/25. doi: 10.1016/j.biomaterials.2012.08.045. PubMed PMID: 22999467; PMCID: PMC3466362.

Abstract: Tissue-engineered constructs designed to treat large cartilage defects or osteoarthritic lesions may be exposed to significant mechanical loading as well as an inflammatory environment upon implantation in an injured or diseased joint. We hypothesized that a three-dimensionally (3D) woven poly(epsilon- caprolactone) (PCL) scaffold seeded with bone marrow-derived mesenchymal stem cells (MSCs) would provide biomimetic mechanical properties in early stages of in vitro culture as the MSCs assembled a functional, cartilaginous extracellular matrix (ECM). We also hypothesized that these properties would be maintained even in the presence of the pro-inflammatory cytokine interleukin-1 (IL-1), which is found at high levels in injured or diseased joints. MSC-seeded 3D woven scaffolds cultured in chondrogenic conditions synthesized a functional ECM rich in collagen and proteoglycan content, reaching an aggregate modulus of ~0.75 MPa within 14 days of culture. However, the presence of pathophysiologically relevant levels of IL-1 limited matrix accumulation and inhibited any increase in mechanical properties over baseline values. On the other hand, the mechanical properties of constructs cultured in chondrogenic conditions for 4 weeks prior to IL-1 exposure were protected from deleterious effects of the cytokine. These findings demonstrate that IL-1 significantly inhibits the chondrogenic development and maturation of MSC-seeded constructs; however, the overall mechanical functionality of the engineered tissue can be preserved through the use of a 3D woven scaffold designed to recreate the mechanical properties of native articular cartilage.

9. Liao IC, Moutos FT, Estes BT, Zhao X, Guilak F. Composite three-dimensional woven scaffolds with interpenetrating network hydrogels to create functional synthetic articular cartilage. Adv Funct Mater. 2013;23(47):5833-9. Epub 2014/03/01. doi: 10.1002/adfm.201300483. PubMed PMID: 24578679; PMCID: PMC3933181.

Abstract: The development of synthetic biomaterials that possess mechanical properties that mimic those of native tissues remains an important challenge to the field of materials. In particular, articular cartilage is a complex nonlinear, viscoelastic, and anisotropic material that exhibits a very low coefficient of friction, allowing it to withstand millions of cycles of joint loading over decades of wear. Here we show that a three-dimensionally woven fiber scaffold that is infiltrated with an interpenetrating network hydrogel can provide a functional biomaterial that provides the load-bearing and tribological properties of native cartilage. An interpenetrating dual-network “tough-gel” consisting of alginate and polyacrylamide was infused into a porous three-dimensionally woven poly(epsilon- caprolactone) fiber scaffold, providing a versatile fiber-reinforced composite structure as a potential acellular or cell-based replacement for cartilage repair.

10. Brunger JM, Huynh NP, Guenther CM, Perez-Pinera P, Moutos FT, Sanchez-Adams J, Gersbach CA, Guilak F. Scaffold-mediated lentiviral transduction for functional tissue engineering of cartilage. Proc Natl Acad Sci U S A. 2014;111(9):E798-806. Epub 2014/02/20. doi: 10.1073/pnas.1321744111. PubMed PMID: 24550481; PMCID: PMC3948308.

Abstract: The ability to develop tissue constructs with matrix composition and biomechanical properties that promote rapid tissue repair or regeneration remains an enduring challenge in musculoskeletal engineering. Current approaches require extensive cell manipulation ex vivo, using exogenous growth factors to drive tissue-specific differentiation, matrix accumulation, and mechanical properties, thus limiting their potential clinical utility. The ability to induce and maintain differentiation of stem cells in situ could bypass these steps and enhance the success of engineering approaches for tissue regeneration. The goal of this study was to generate a self-contained bioactive scaffold capable of mediating stem cell differentiation and formation of a cartilaginous extracellular matrix (ECM) using a lentivirus- based method. We first showed that poly-L-lysine could immobilize lentivirus to poly(epsilon- caprolactone) films and facilitate human mesenchymal stem cell (hMSC) transduction. We then demonstrated that scaffold-mediated gene delivery of transforming growth factor beta3 (TGF-beta3), using a 3D woven poly(epsilon-caprolactone) scaffold, induced robust cartilaginous ECM formation by hMSCs. Chondrogenesis induced by scaffold-mediated gene delivery was as effective as traditional differentiation protocols involving medium supplementation with TGF-beta3, as assessed by gene expression, biochemical, and biomechanical analyses. Using lentiviral vectors immobilized on a biomechanically functional scaffold, we have developed a system to achieve sustained transgene expression and ECM formation by hMSCs. This method opens new avenues in the development of bioactive implants that circumvent the need for ex vivo tissue generation by enabling the long-term goal of in situ tissue engineering.

11. Glass KA, Link JM, Brunger JM, Moutos FT, Gersbach CA, Guilak F. Tissue-engineered cartilage with inducible and tunable immunomodulatory properties. Biomaterials. 2014;35(22):5921-31. Epub 2014/04/29. doi: 10.1016/j.biomaterials.2014.03.073. PubMed PMID: 24767790; PMCID: PMC4047672.

Abstract: The pathogenesis of osteoarthritis is mediated in part by inflammatory cytokines including interleukin- 1 (IL-1), which promote degradation of articular cartilage and prevent human mesenchymal stem cell (MSC) chondrogenesis. In this study, we combined gene therapy and functional tissue engineering to develop engineered cartilage with immunomodulatory properties that allow chondrogenesis in the presence of pathologic levels of IL-1 by inducing overexpression of IL-1 receptor antagonist (IL-1Ra) in MSCs via scaffold-mediated lentiviral gene delivery. A doxycycline-inducible vector was used to transduce MSCs in monolayer or within 3D woven PCL scaffolds to enable tunable IL-1Ra production. In the presence of IL-1, IL-1Ra-expressing engineered cartilage produced cartilage-specific extracellular matrix, while resisting IL-1-induced upregulation of matrix metalloproteinases and maintaining mechanical properties similar to native articular cartilage. The ability of functional engineered cartilage to deliver tunable anti-inflammatory cytokines to the joint may enhance the long-term success of therapies for cartilage injuries or osteoarthritis.

12. Moutos FT, Glass KA, Compton SA, Ross AK, Gersbach CA, Guilak F, Estes BT. Anatomically shaped tissue-engineered cartilage with tunable and inducible anticytokine delivery for biological joint resurfacing. Proc Natl Acad Sci U S A. 2016;113(31):E4513-22. Epub 2016/07/20. doi: 10.1073/pnas.1601639113. PubMed PMID: 27432980; PMCID: PMC4978289.

Abstract: Biological resurfacing of entire articular surfaces represents an important but challenging strategy for treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chondrogenesis and induce degradation of native and engineered cartilage. The goal of this study was to use adult stem cells to engineer anatomically shaped, functional cartilage constructs capable of tunable and inducible expression of antiinflammatory molecules, specifically IL-1 receptor antagonist (IL-1Ra). Large (22-mm-diameter) hemispherical scaffolds were fabricated from 3D woven poly(epsilon-caprolactone) (PCL) fibers into two different configurations and seeded with human adipose-derived stem cells (ASCs). Doxycycline (dox)-inducible lentiviral vectors containing eGFP or IL-1Ra transgenes were immobilized to the PCL to transduce ASCs upon seeding, and constructs were cultured in chondrogenic conditions for 28 d. Constructs showed biomimetic cartilage properties and uniform tissue growth while maintaining their anatomic shape throughout culture. IL-1Ra-expressing constructs produced nearly 1 microg/mL of IL-1Ra upon controlled induction with dox. Treatment with IL-1 significantly increased matrix metalloprotease activity in the conditioned media of eGFP-expressing constructs but not in IL-1Ra-expressing constructs. Our findings show that advanced textile manufacturing combined with scaffold-mediated gene delivery can be used to tissue engineer large anatomically shaped cartilage constructs that possess controlled delivery of anticytokine therapy. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function.

13. Recha-Sancho L, Moutos FT, Abella J, Guilak F, Semino CE. Dedifferentiated Human Articular Chondrocytes Redifferentiate to a Cartilage-Like Tissue Phenotype in a Poly(epsilon- Caprolactone)/Self- Assembling Peptide Composite Scaffold. Materials (Basel). 2016;9(6). Epub 2016/06/17. doi: 10.3390/ma9060472. PubMed PMID: 28773609; PMCID: PMC5456812.

Abstract: Adult articular cartilage has a limited capacity for growth and regeneration and, with injury, new cellular or biomaterial-based therapeutic platforms are required to promote repair. Tissue engineering aims to produce cartilage-like tissues that recreate the complex mechanical and biological properties found in vivo. In this study, a unique composite scaffold was developed by infiltrating a three- dimensional (3D) woven microfiber poly (epsilon-caprolactone) (PCL) scaffold with the RAD16-I self- assembling nanofibers to obtain multi-scale functional and biomimetic tissue-engineered constructs. The scaffold was seeded with expanded dedifferentiated human articular chondrocytes and cultured for four weeks in control and chondrogenic growth conditions. The composite constructs were compared to control constructs obtained by culturing cells with 3D woven PCL scaffolds or RAD16-I independently. High viability and homogeneous cell distribution were observed in all three scaffolds used during the term of the culture. Moreover, gene and protein expression profiles revealed that chondrogenic markers were favored in the presence of RAD16-I peptide (PCL/RAD composite or alone) under chondrogenic induction conditions. Further, constructs displayed positive staining for toluidine blue, indicating the presence of synthesized proteoglycans. Finally, mechanical testing showed that constructs containing the PCL scaffold maintained the initial shape and viscoelastic behavior throughout the culture period, while constructs with RAD16-I scaffold alone contracted during culture time into a stiffer and compacted structure. Altogether, these results suggest that this new composite scaffold provides important mechanical requirements for a cartilage replacement, while providing a biomimetic microenvironment to re-establish the chondrogenic phenotype of human expanded articular chondrocytes.

14. Huynh NPT, Brunger JM, Gloss CC, Moutos FT, Gersbach CA, Guilak F. Genetic Engineering of Mesenchymal Stem Cells for Differential Matrix Deposition on 3D Woven Scaffolds. Tissue Eng Part A. 2018;24(19-20):1531-44. Epub 2018/05/15. doi: 10.1089/ten.TEA.2017.0510. PubMed PMID: 29756533; PMCID: PMC6198766.

Abstract: Tissue engineering approaches for the repair of osteochondral defects using biomaterial scaffolds and stem cells have remained challenging due to the inherent complexities of inducing cartilage-like matrix and bone-like matrix within the same local environment. Members of the transforming growth factor beta (TGFbeta) family have been extensively utilized in the engineering of skeletal tissues, but have distinct effects on chondrogenic and osteogenic differentiation of progenitor cells. The goal of this study was to develop a method to direct human bone marrow-derived mesenchymal stem cells (MSCs) to deposit either mineralized matrix or a cartilaginous matrix rich in glycosaminoglycan and type II collagen within the same biochemical environment. This differential induction was performed by culturing cells on engineered three-dimensionally woven poly(varepsilon- caprolactone) (PCL) scaffolds in a chondrogenic environment for cartilage-like matrix production while inhibiting TGFbeta3 signaling through Mothers against DPP homolog 3 (SMAD3) knockdown, in combination with overexpressing RUNX2, to achieve mineralization. The highest levels of mineral deposition and alkaline phosphatase activity were observed on scaffolds with genetically engineered MSCs and exhibited a synergistic effect in response to SMAD3 knockdown and RUNX2 expression. Meanwhile, unmodified MSCs on PCL scaffolds exhibited accumulation of an extracellular matrix rich in glycosaminoglycan and type II collagen in the same biochemical environment. This ability to derive differential matrix deposition in a single culture condition opens new avenues for developing complex tissue replacements for chondral or osteochondral defects.

15. Moffat KL, Goon K, Moutos FT, Estes BT, Oswald SJ, Zhao X, Guilak F. Composite Cellularized Structures Created from an Interpenetrating Polymer Network Hydrogel Reinforced by a 3D Woven Scaffold. Macromol Biosci. 2018;18(10):e1800140. Epub 2018/07/25. doi: 10.1002/mabi.201800140. PubMed PMID: 30040175; PMCID: PMC6687075.

Abstract: Biomaterial scaffolds play multiple roles in cartilage tissue engineering, including controlling architecture of newly formed tissue while facilitating growth of embedded cells and simultaneously providing functional properties to withstand the mechanical environment within the native joint. In particular, hydrogels-with high water content and desirable transport properties-while highly conducive to chondrogenesis, often lack functional mechanical properties. In this regard, interpenetrating polymer network (IPN) hydrogels can provide mechanical toughness greatly exceeding that of individual components; however, many IPN materials are not biocompatible for cell encapsulation. In this study, an agarose and poly(ethylene) glycol IPN hydrogel is seeded with human mesenchymal stem cells (MSCs). Results show high viability of MSCs within the IPN hydrogel, with improved mechanical properties compared to constructs comprised of individual components. These properties are further strengthened by integrating the hydrogel with a 3D woven structure. The resulting fiber-reinforced hydrogels display functional macroscopic mechanical properties mimicking those of native articular cartilage, while providing a local microenvironment that supports cellular viability and function. These findings suggest that a fiber-reinforced IPN hydrogel can support stem cell chondrogenesis while allowing for significantly enhanced, complex mechanical properties at multiple scales as compared to individual hydrogel or fiber components.

16. Venkatesan JK, Moutos FT, Rey-Rico A, Estes BT, Frisch J, Schmitt G, Madry H, Guilak F, Cucchiarini M. Chondrogenic Differentiation Processes in Human Bone-Marrow Aspirates Seeded in Three- Dimensional-Woven Poly(varepsilon-Caprolactone) Scaffolds Enhanced by Recombinant Adeno- Associated Virus-Mediated SOX9 Gene Transfer. Hum Gene Ther. 2018;29(11):1277-86. Epub 2018/05/03. doi: 10.1089/hum.2017.165. PubMed PMID: 29717624; PMCID: PMC6247986.

Abstract: Combining gene therapy approaches with tissue engineering procedures is an active area of translational research for the effective treatment of articular cartilage lesions, especially to target chondrogenic progenitor cells such as those derived from the bone marrow. This study evaluated the effect of genetically modifying concentrated human mesenchymal stem cells from bone marrow to induce chondrogenesis by recombinant adeno-associated virus (rAAV) vector gene transfer of the sex- determining region Y-type high-mobility group box 9 (SOX9) factor upon seeding in three-dimensional- woven poly(varepsilon-caprolactone; PCL) scaffolds that provide mechanical properties mimicking those of native articular cartilage. Prolonged, effective SOX9 expression was reported in the constructs for at least 21 days, the longest time point evaluated, leading to enhanced metabolic and chondrogenic activities relative to the control conditions (reporter lacZ gene transfer or absence of vector treatment) but without affecting the proliferative activities in the samples. The application of the rAAV SOX9 vector also prevented undesirable hypertrophic and terminal differentiation in the seeded concentrates. As bone marrow is readily accessible during surgery, such findings reveal the therapeutic potential of providing rAAV-modified marrow concentrates within three-dimensional-woven PCL scaffolds for repair of focal cartilage lesions.

17. Larson BL, Yu SN, Park H, Estes BT, Moutos FT, Bloomquist CJ, Wu PB, Welter JF, Langer R, Guilak F, Freed LE. Chondrogenic, hypertrophic, and osteochondral differentiation of human mesenchymal stem cells on three-dimensionally woven scaffolds. J Tissue Eng Regen Med. 2019;13(8):1453-65. Epub 2019/05/23. doi: 10.1002/term.2899. PubMed PMID: 31115161; PMCID: PMC6715532.

Abstract: The development of mechanically functional cartilage and bone tissue constructs of clinically relevant size, as well as their integration with native tissues, remains an important challenge for regenerative medicine. The objective of this study was to assess adult human mesenchymal stem cells (MSCs) in large, three-dimensionally woven poly(epsilon- caprolactone; PCL) scaffolds in proximity to viable bone, both in a nude rat subcutaneous pouch model and under simulated conditions in vitro. In Study I, various scaffold permutations-PCL alone, PCL-bone, “point-of-care” seeded MSC-PCL-bone, and chondrogenically precultured Ch-MSC-PCL-bone constructs-were implanted in a dorsal, ectopic pouch in a nude rat. After 8 weeks, only cells in the Ch-MSC-PCL constructs exhibited both chondrogenic and osteogenic gene expression profiles. Notably, although both tissue profiles were present, constructs that had been chondrogenically precultured prior to implantation showed a loss of glycosaminoglycan (GAG) as well as the presence of mineralization along with the formation of trabecula-like structures. In Study II of the study, the GAG loss and mineralization observed in Study I in vivo were recapitulated in vitro by the presence of either nearby bone or osteogenic culture medium additives but were prevented by a continued presence of chondrogenic medium additives. These data suggest conditions under which adult human stem cells in combination with polymer scaffolds synthesize functional and phenotypically distinct tissues based on the environmental conditions and highlight the potential influence that paracrine factors from adjacent bone may have on MSC fate, once implanted in vivo for chondral or osteochondral repair.

18. Sennett ML, Friedman JM, Ashley BS, Stoeckl BD, Patel JM, Alini M, Cucchiarini M, Eglin D, Madry H, Mata A, Semino C, Stoddart MJ, Johnstone B, Moutos FT, Estes BT, Guilak F, Mauck RL, Dodge GR. Long term outcomes of biomaterial-mediated repair of focal cartilage defects in a large animal model. Eur Cell Mater. 2021;41:40-51. Epub 2021/01/08. doi:10.22203/eCM.v041a04. PubMed PMID: 33411938; PMCID: PMC8626827.

Abstract: Articular cartilage has unique load-bearing properties but has minimal capacity for intrinsic repair. Here, we used three-dimensional weaving, additive manufacturing, and autologous mesenchymal stem cells to create a tissue- engineered, bicomponent implant to restore hip function in a canine hip osteoarthritis model. This resorbable implant was specifically designed to function mechanically from the time of repair and to biologically integrate with native tissues for long-term restoration. A massive osteochondral lesion was created in the hip of skeletally mature hounds and repaired with the implant or left empty (control). Longitudinal outcome measures over 6 months demonstrated that the implant dogs returned to normal preoperative values of pain and function. Anatomical structure and functional biomechanical properties were also restored in the implanted dogs. Control animals never returned to normal and exhibited structurally deficient repair. This study provides clinically relevant evidence that the bicomponent implant may be a potential therapy for moderate hip osteoarthritis.

19. Estes BT, Enomoto M, Moutos FT, Carson MA, Toth JM, Eggert P, Stallrich J, Willard VP, Veis DJ, Little D, Guilak F, Lascelles BDX. Biological resurfacing in a canine model of hip osteoarthritis. Sci Adv. 2021;7(38):eabi5918. Epub 2021/09/16. doi: 10.1126/sciadv.abi5918. PubMed PMID: 34524840; PMCID: PMC8443182.

Abstract: Articular cartilage has unique load-bearing properties but has minimal capacity for intrinsic repair. Here, we used three-dimensional weaving, additive manufacturing, and autologous mesenchymal stem cells to create a tissue- engineered, bicomponent implant to restore hip function in a canine hip osteoarthritis model. This resorbable implant was specifically designed to function mechanically from the time of repair and to biologically integrate with native tissues for long-term restoration. A massive osteochondral lesion was created in the hip of skeletally mature hounds and repaired with the implant or left empty (control). Longitudinal outcome measures over 6 months demonstrated that the implant dogs returned to normal preoperative values of pain and function. Anatomical structure and functional biomechanical properties were also restored in the implanted dogs. Control animals never returned to normal and exhibited structurally deficient repair. This study provides clinically relevant evidence that the bicomponent implant may be a potential therapy for moderate hip osteoarthritis.

20. Choi YR, Collins KH, Springer LE, Pferdehirt L, Ross AK, Wu CL, Moutos FT, Harasymowicz NS, Brunger JM, Pham CTN, Guilak F. A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery. Sci Adv. 2021;7(36):eabj1414. Epub 2021/09/14. doi: 10.1126/sciadv.abj1414. PubMed PMID: 34516920; PMCID: PMC8442875.

Abstract: Biologic drug therapies are increasingly used for inflammatory diseases such as rheumatoid arthritis (RA), but they suppress the immune system if administered continuously at high doses, which may cause significant adverse effects. Here, we developed a bioartificial drug delivery system that senses changing levels of endogenous inflammatory cytokines to trigger a proportional therapeutic responses as a means of more appropriately dosing anti- cytokine therapies. Using CRISPR-Cas9 genome editing, we engineered stem cells containing a synthetic gene circuit expressing a biologic drug to inhibit inflammatory cytokines in an autoregulated, feedback-controlled manner in response to activation of the endogenous chemokine (C-C) motif ligand 2 (Ccl2) promoter. When tissue-engineered into stable cartilaginous constructs and implanted subcutaneously, the bioengineered cells were rapidly activated in response to inflammatory stimuli, automatically turned off when stimulation was removed, and could be reactivated upon serial challenges with inflammatory triggers. Using the murine K/BxN model of inflammatory arthritis, we showed that bioengineered implants not only provided a non-invasive luminescent readout of disease severity, but also significantly mitigated disease severity as measured by joint pain, structural damage, and systemic and local inflammation. More importantly, therapeutic implants completely prevented increased pain sensitivity and bone erosions, a feat not currently achievable by any clinically available disease-modifying drugs. The implants provided greater therapeutic benefit as compared to several standard of care treatments. The combination of tissue engineering and synthetic biology promises a range of potential applications for treating chronic diseases via custom-designed cells that express therapeutic transgenes in response to dynamically changing biological signals.

21. Guilak F, Estes BT, Moutos FT. Functional tissue engineering of articular cartilage for biological joint resurfacing-The 2021 Elizabeth Winston Lanier Kappa Delta Award. J Orthop Res. 2022;40(8):1721-34. Epub 2021/11/24. doi: 10.1002/jor.25223. PubMed PMID: 34812518; PMCID: PMC9124734.

Abstract: Biological resurfacing of entire articular surfaces represents a challenging strategy for the treatment of cartilage degeneration that occurs in osteoarthritis. Not only does this approach require anatomically sized and functional engineered cartilage, but the inflammatory environment within an arthritic joint may also inhibit chon- drogenesis and induce degradation of native and engineered cartilage. Here, we present the culmination of multiple avenues of interdisciplinary research leading to the development and testing of bioartificial cartilage for tissue-engineered resurfa- cing of the hip joint. The work is based on a novel three-dimensional weaving technology that is infiltrated with specific bioinductive materials and/or genetically- engineered stem cells. A variety of design approaches have been tested in vitro, showing biomimetic cartilage-like properties as well as the capability for long-term tunable and inducible drug delivery. Importantly, these cartilage constructs have the potential to provide mechanical functionality immediately upon implantation, as they will need to replace a majority, if not the entire joint surface to restore function. To date, these approaches have shown excellent preclinical success in a variety of animal studies, including the resurfacing of a large osteochondral defect in the canine hip, and are now well-poised for clinical translation.