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Cellular senescence is a state of terminal growth arrest associated with the upregulation of different cell cycle inhibitors, mainly p16 and p21, structural and metabolic alterations, chronic DNA damage responses, and a hypersecretory state known as the senescence-associated secretory phenotype (SASP). The SASP is the major mediator of the paracrine effects of senescent cells in their tissue microenvironment and of various local and systemic biological functions. In this Review, we discuss the composition, dynamics and heterogeneity of the SASP as well as the mechanisms underlying its induction and regulation. We describe the various biological properties of the SASP, its beneficial and detrimental effects in different physiological and pathological settings, and its impact on overall health span. Finally, we discuss the use of the SASP as a biomarker and of SASP inhibitors as senomorphic interventions to treat cancer and other age-related conditions.

The interleukin-4 (IL-4) cytokine plays a critical role in modulating immune homeostasis. Although there is great interest in harnessing this cytokine as a therapeutic in natural or engineered formats, the clinical potential of native IL-4 is limited by its instability and pleiotropic actions. Here, we design IL-4 cytokine mimetics (denoted Neo-4) based on a de novo engineered IL-2 mimetic scaffold and demonstrate that these cytokines can recapitulate physiological functions of IL-4 in cellular and animal models. In contrast with natural IL-4, Neo-4 is hyperstable and signals exclusively through the type I IL-4 receptor complex, providing previously inaccessible insights into differential IL-4 signaling through type I versus type II receptors. Because of their hyperstability, our computationally designed mimetics can directly incorporate into sophisticated biomaterials that require heat processing, such as three-dimensional-printed scaffolds. Neo-4 should be broadly useful for interrogating IL-4 biology, and the design workflow will inform targeted cytokine therapeutic development.

Vascularized composite allotransplantation (VCA) is a restorative option for patients suffering from severe tissue defects not amenable to conventional reconstruction. However, the toxicities associated with life-long multidrug immunosuppression to enable allograft survival and induce immune tolerance largely limit the broader application of VCA. Here, we investigate the potential of targeted immunomodulation using CTLA4-Ig combined with a biological porcine-derived extracellular matrix (ECM) scaffold that elicits a pro-regenerative Th2 response to promote allograft survival and regulate the inflammatory microenvironment in a stringent mouse orthotopic hind limb transplantation model (BALB/c to C57BL/6). The median allograft survival time (MST) increased significantly from 15.0 to 24.5 days (P= 0.0037; Mantel–Cox test) after adding ECM to the CTLA4-Ig regimen. Characterization of the immune infiltration shows a pro-regenerative phenotype prevails over those associated with inflammation and rejection including macrophages (F4/80hi+CD206hi+MHCIIlow), eosinophils (F4/80lowSiglec-F+), and T helper 2 (Th2) T cells (CD4+IL-4+). This was accompanied by an increased expression of genes associated with a Type 2 polarized immune state such as Il4, Ccl24, Arg1and Ym1within the graft. Furthermore, when ECM was applied along with a clinically relevant combination of CTLA4-Ig and Rapamycin, allograft survival was prolonged from 33.0 to 72.5 days (P= 0.0067; Mantel–Cox test). These studies implicate the clinical exploration of combined regimens involving local application of pro-regenerative, immunomodulatory biomaterials in surgical wound sites with targeted co-stimulatory blockade to reduce adverse effects of immunosuppression and enhance graft survival in VCA.

Cellular senescence is a state of permanent growth arrest that plays an important role in wound healing, tissue fibrosis, and tumor suppression. Despite senescent cells’ (SnCs) pathological role and therapeutic interest, their phenotype in vivo remains poorly defined. Here, we developed an in vivo–derived senescence signature (SenSig) using a foreign body response–driven fibrosis model in a p16-CreERT2;Ai14reporter mouse. We identified pericytes and “cartilage-like” fibroblasts as senescent and defined cell type–specific senescence-associated secretory phenotypes (SASPs). Transfer learning and senescence scoring identified these two SnC populations along with endothelial and epithelial SnCs in new and publicly available murine and human data single-cell RNA sequencing (scRNAseq) datasets from diverse pathologies. Signaling analysis uncovered crosstalk between SnCs and myeloid cells via an IL34–CSF1R–TGFβR signaling axis, contributing to tissue balance of vascularization and matrix production. Overall, our study provides a senescence signature and a computational approach that may be broadly applied to identify SnC transcriptional profiles and SASP factors in wound healing, aging, and other pathologies.

The foreign body response (FBR) remains a clinical challenge in the field of biomaterials due to its ability to elicit a chronic and sustained immune response. Modulating the immune response to materials is a modern paradigm in tissue engineering to enhance repair while limiting fibrous encapsulation and implant isolation. Though the classical mediators of the FBR are well-characterized, recent studies highlight that our understanding of the cell types that shape the FBR may be incomplete. In this review, we discuss the emerging role of T cells, stromal-immune cell interactions, and senescent cells in the biomaterial response, particularly to synthetic materials. We emphasize future studies that will deepen the field's understanding of these cell types in the FBR, with the goal of identifying therapeutic targets that will improve implant integration. Finally, we briefly review several considerations that may influence our understanding of the FBR in humans, including rodent models, aging, gut microbiota, and sex differences. A better understanding of the heterogeneous host cell response during the FBR can enable the design and development of immunomodulatory materials that favor healing.

The immune system plays a critical role in directing tissue repair and regeneration outcomes. Tissue engineering technologies that are designed to promote new tissue growth will therefore be impacted by immune factors that are present in patients both locally at the site of intervention and systemically. The immune state of patients can be influenced by many factors, including infection, nutrition, and other disease comorbidities. As a result, the immune state is highly variable and may be a source of variability in tissue-engineered products in the clinic, which is not found in preclinical models. In this review, we will summarize key immune cells and evidence of their activity in tissue repair and potential in tissue engineering systems. We also discuss how clinical translation of tissue engineering strategies, in particular stem cells, helped elucidate the importance of the immune system. With increased understanding of the immune system's role in repair and tissue engineering systems, it will likely become a therapeutic target and component of future therapies.

Objective To determine performance and repair kinetics of the ChonDux hydrogel scaffold for treating focal articular cartilage defects in the knee over 24 months.Design This assessor-blinded trial evaluates ChonDux hydrogel scaffold implantation in combination with microfracture in 18 patients across 6 sites. Male and female patients 18 to 65 years of age with full-thickness femoral condyle defects 2 to 4 cm2in area were enrolled. Eligible patients received ChonDux treatment followed by rehabilitation. Defect volume fill was evaluated after 3, 6 (primary outcome), 12, 18, and 24 months by assessor blinded magnetic resonance imaging (MRI) analysis. Secondary outcomes were T2-weighted MRI relaxation time and patient surveys via visual analogue scale (VAS) pain and International Knee Documentation Committee (IKDC) knee function scoring.Results ChonDux maintained durable tissue restoration over 24 months with final defect percent fill of 94.2% ± 16.3% and no significant loss of fill volume at any time points. Tissues treated with ChonDux maintained T2 relaxation times similar to uninjured cartilage between 12 and 24 months. VAS pain scoring decreased between 1 and 6 weeks, and IKDC knee function scores improved by approximately 30.1 with ChonDux over 24 months.Conclusion ChonDux treatment is a safe adjunct to microfracture therapy and promotes stable restoration of full thickness articular cartilage defects for at least 24 months.

Fibrosis is a pathological accumulation of excessive collagen that underlies many of the most common diseases, representing dysfunction of the essential processes of normal tissue healing. Fibrosis research aims to limit this response without ameliorating the essential role of fibrogenesis in organ function. However, the absence of a realistic in vitromodel has hindered investigation into mechanisms and potential interventions because the standard 2D monolayer culture of fibroblasts has limited applicability. We sought to develop and optimize fibrosis spheroids: a scaffold-free three-dimensional human fibroblast-macrophage spheroid system representing an improved benchtop model of human fibrosis. We created, characterized and optimized human fibroblast-only spheroids, demonstrating increased collagen deposition compared to monolayer fibroblasts, while spheroids larger than 300 μm suffered from progressively increasing apoptosis. Next, we improved the spheroid system with the addition of human macrophages to more precisely recapitulate the environment during fibrogenesis, creating a hybrid spheroid system with different ratios of fibroblasts and macrophages ranging from 2 : 1 to 64 : 1. We found that in the hybrid spheroids (particularly the 16 : 1 [F16] ratio) more fibroblasts were activated, with greater macrophage polarization towards a pro-inflammatory M1 phenotype. Hybrid spheroids containing higher ratios of macrophages showed greater macrophage heterogeneity and less fibrogenesis, while low macrophage ratios limited macrophage-induced effects and yielded less collagen deposition. The F16 group also had the highest expression levels of fibrosis-related genes (Col-1a1, Col-3a1and TGF-β) and inflammation-related genes (TNF, IL1βand IL6). IF staining demonstrated that F16 spheroids had the highest levels of αSMA, collagen-1 and collagen-3 deposition among all groups as well as formation of a dense collagen rim surrounding the spheroid. Future studies exploring the greater fibrotic activity of F16 spheroids may provide new mechanistic insights into diseases involving excessive fibrotic activity. Microtissue fibrosis models capable of achieving greater clinical fidelity have the potential to combine the relevance of animal models with the scale, cost and throughput of in vitrotesting.

During the foreign body response, immune cells are metabolically rewired after exposure to breakdown products of various biomaterials, including polylactide (PLA) and polyethylene. Particles of polyethylene interact with Toll-like receptor 4 on macrophages, resulting in increased oxygen consumption that forms reactive oxygen species at complex I of the mitochondrial electron transport chain (mETC). However, PLA degradation products bind to monocarboxylate transporters for downstream signaling with elevated oxygen consumption rates, whose functional implication is unclear and remains inferred from cellular responses to polyethylene biomaterials. By chemically probing the function of the mETC, we show that proinflammatory macrophages activated by exposure to amorphous PLA (aPLA) breakdown products rely on mitochondrial respiration for ATP production independent of oxygen consumption rates. In contrast, macrophages activated by semicrystalline PLA (cPLA) breakdown products exhibit a metabolic phenotype wherein ATP levels are unaffected by changing oxygen consumption rates. In subcutaneous implants, the incorporation of metformin in aPLA or cPLA to chemically inhibit complex I did not effectively modulate the proinflammatory response to biomaterials, suggesting that PLA degradation products elicit a distinct metabolic program, thus providing an alternative perspective on the role of mitochondrial respiration in the inflammatory response to biomaterials.

Triggering shape-memory functionality under clinical hyperthermia temperatures could enable the control and actuation of shape-memory systems in clinical practice. For this purpose, we developed light-inducible shape-memory microparticles composed of a poly(d,l-lactic acid) (PDLLA) matrix encapsulating gold nanoparticles (Au@PDLLA hybrid microparticles). This shape-memory polymeric system for the first time demonstrates the capability of maintaining an anisotropic shape at body temperature with triggered shape-memory effect back to a spherical shape at a narrow temperature range above body temperature with a proper shape recovery speed (37 < T< 45 °C). We applied a modified film-stretching processing method with carefully controlled stretching temperature to enable shape memory and anisotropy in these micron-sized particles. Accordingly, we achieved purely entanglement-based shape-memory response without chemical cross-links in the miniaturized shape-memory system. Furthermore, these shape-memory microparticles exhibited light-induced spatiotemporal control of their shape recovery using a laser to trigger the photothermal heating of doped gold nanoparticles. This shape-memory system is composed of biocompatible components and exhibits spatiotemporal controllability of its properties, demonstrating a potential for various biomedical applications, such as tuning macrophage phagocytosis as demonstrated in this study.

Composite biomaterials comprising polylactide (PLA) and hydroxyapatite (HA) are applied in bone, cartilage and dental regenerative medicine, where HA confers osteoconductive properties. However, after surgical implantation, adverse immune responses to these composites can occur, which have been attributed to size and morphology of HA particles. Approaches to effectively modulate these adverse immune responses have not been described. PLA degradation products have been shown to alter immune cell metabolism (immunometabolism), which drives the inflammatory response. Accordingly, to modulate the inflammatory response to composite biomaterials, inhibitors were incorporated into composites comprised of amorphous PLA (aPLA) and HA (aPLA+HA) to regulate glycolytic flux. Inhibition at specific steps in glycolysis reduced proinflammatory (CD86+CD206-) and increased pro-regenerative (CD206+) immune cell populations around implanted aPLA+HA. Notably, neutrophil and dendritic cell (DC) numbers along with proinflammatory monocyte and macrophage populations were decreased, and Arginase 1 expression among DCs was increased. Targeting immunometabolism to control the proinflammatory response to biomaterial composites, thereby creating a pro-regenerative microenvironment, is a significant advance in tissue engineering where immunomodulation enhances osseointegration and angiogenesis, which could lead to improved bone regeneration.

The immune system has evolved as a powerful tool for our body to combat infections, and is being engineered for new treatments in cancer and autoimmune disease. More recently, the complex role of the immune system is being recognized in tissue repair, regenerative medicine and biomaterial responses. From these combined interests, the field of immunoengineering is rapidly growing. However, bridging immunology with engineering poses numerous challenges including the biological complexity, language of immunology and accurately leveraging the powerful techniques of immunology to new applications. Elucidating the identity and function of immune cell populations responding to engineering systems will be required for continued advancement. Multi-color flow cytometry is a central technique used by immunologists for this purpose that requires careful control of variables, data acquisition, and interpretation. Here, we present methods for multi-color flow cytometry experimental design and analysis focused on characterizing the scaffold immune microenvironment in regenerative medicine research.

Objective: Develop a drug-eluting stent construct with a reliable drug-release profile and adequate mechanically stability for a trial in a small animal model of laryngotracheal stenosis (LTS), a debilitating pathologic narrowing of the airway leading to significant shortness of breath. Methods: Biodegradable, biocompatible stents containing 1.0% rapamycin made of PLLA-PCL (70% Poly-l-Lactide and 30% Polycaprolactone blend) and 50 : 50 PDLGA (Poly(dl-lactide-co-glycolide)) were compared. Mechanical strength testing and drug elution rates using high performance liquid chromatography analysis (HPLC) was assessed. Next, efficacy of stent elution on LTS derived scar fibroblasts. Finally, stents were placed in situin an LTS mouse model. Results: The PLLA-PCL stent construct exhibited greater mechanical strength compared to the PDLGA stent over a 4-week period (Young's Modulus (PLLA-PCL) = 13.82; Young's Modulus (PDLGA) = 4.015). Moreover, the PLLA-PCL stent showed a reliable rapamycin release profile for 6 weeks (30% elution for PLLA-PCL stents compared to <1% elution for PDLGA). Collagen 1 (p< 0.05) and fibroblast cell proliferation were decreased in vitrowhen treated with the rapamycin stent. In vivo, the rapamycin stent reduced lamina propria thickness (p< 0.05) and collagen 1(p< 0.05), collagen 3, TGF-B (p< 0.05) and a-SMA (p< 0.05). Conclusions: The PLLA-PCL construct demonstrated superior mechanical strength and greater drug elution compared to PDLGA stents. We demonstrated the feasibility of testing this drug-eluting stent in vivo, showing that the rapamycin-eluting stent treats fibrosis. To our knowledge this is the first study to deploy a drug-eluting stent to treat tracheal pathology in an animal model. Optimization of a rapamycin-eluting PLLA-PCL stent for translational investigation will lead to improved treatment strategies of LTS.

Severe damage to the ocular surface can result in limbal stem cell (LSC) deficiency, which contributes to loss of corneal clarity, potential vision loss, chronic pain, photophobia, and keratoplasty failure. Human amniotic membrane (AM) is the most effective substrate for LSC transplantation to treat patients with LSC deficiency. However, the widespread use of the AM in the clinic remains a challenge because of the high cost for preserving freshly prepared AM and the weak mechanical strength of lyophilized AM. Here, we developed a novel composite membrane consisting of an electrospun bioabsorbable polymer fiber mesh bonded to a decellularized AM (dAM) sheet through interfacial conjugation. This membrane engineering approach drastically improved the tensile property and toughness of dAM, preserved similar levels of bioactivities as the dAM itself in supporting LSC attachment, growth, and maintenance, and retained significant anti-inflammatory capacity. These results demonstrate that the lyophilized nanofiber–dAM composite membrane offers superior mechanical properties for easy handling and suturing to the dAM, while presenting biochemical cues and basement membrane structure to facilitate LSC transplantation. This composite membrane exhibits major advantages for clinical applications in treating soft tissue damage and LSC deficiency.

The immune system mediates tissue growth and homeostasis and is the first responder to injury or biomaterial implantation. Recently, it has been appreciated that immune cells play a critical role in wound healing and tissue repair and should thus be considered potentially beneficial, particularly in the context of scaffolds for regenerative medicine. In this study, we present a flow cytometric analysis of cellular recruitment to tissue-derived extracellular matrix scaffolds, where we quantitatively describe the infiltration and polarization of several immune subtypes, including macrophages, dendritic cells, neutrophils, monocytes, T cells, and B cells. We define a specific scaffold-associated macrophage (SAM) that expresses CD11b+F4/80+CD11c+/−CD206hiCD86+MHCII+that are characteristic of an M2-like cell (CD206hi) with high antigen presentation capabilities (MHCII+). Adaptive immune cells tightly regulate the phenotype of a mature SAM. These studies provide a foundation for detailed characterization of the scaffold immune microenvironment of a given biomaterial scaffold to determine the effect of scaffold changes on immune response and subsequent therapeutic outcome of that material.

Senescent cells (SnCs) accumulate in many vertebrate tissues with age and contribute to age-related pathologies, presumably through their secretion of factors contributing to the senescence-associated secretory phenotype (SASP). Removal of SnCs delays several pathologies and increases healthy lifespan. Aging and trauma are risk factors for the development of osteoarthritis (OA), a chronic disease characterized by degeneration of articular cartilage leading to pain and physical disability. Senescent chondrocytes are found in cartilage tissue isolated from patients undergoing joint replacement surgery, yet their role in disease pathogenesis is unknown. To test the idea that SnCs might play a causative role in OA, we used the p16-3MR transgenic mouse, which harbors a p16INK4a(Cdkn2a) promoter driving the expression of a fusion protein containing synthetic Renilla luciferase and monomeric red fluorescent protein domains, as well as a truncated form of herpes simplex virus 1 thymidine kinase (HSV-TK). This mouse strain allowed us to selectively follow and remove SnCs after anterior cruciate ligament transection (ACLT). We found that SnCs accumulated in the articular cartilage and synovium after ACLT, and selective elimination of these cells attenuated the development of post-traumatic OA, reduced pain and increased cartilage development. Intra-articular injection of a senolytic molecule that selectively killed SnCs validated these results in transgenic, non-transgenic and aged mice. Selective removal of the SnCs from in vitro cultures of chondrocytes isolated from patients with OA undergoing total knee replacement decreased expression of senescent and inflammatory markers while also increasing expression of cartilage tissue extracellular matrix proteins. Collectively, these findings support the use of SnCs as a therapeutic target for treating degenerative joint disease.

Brown adipose tissue (BAT) has a unique capacity to expend calories by decoupling energy expenditure from ATP production, therefore BAT could realize therapeutic potential to treat metabolic diseases such as obesity and type 2 diabetes. Recent studies have investigated markers and function of native BAT, however, successful therapies will rely on methods that supplement the small existing pool of brown adipocytes in adult humans. In this study, we engineered BAT from both human and rat adipose precursors and determined whether these ex vivoconstructs could mimic in vivotissue form and metabolic function. Adipose-derived stem cells (ASCs) were isolated from several sources, human white adipose tissue (WAT), rat WAT, and rat BAT, then differentiated toward both white and brown adipogenic lineages in two-dimensional and three-dimensional (3D) culture conditions. ASCs derived from WAT were successfully differentiated in 3D poly(ethylene glycol) hydrogels into mature adipocytes with BAT phenotype and function, including high uncoupling protein 1 (UCP1) mRNA and protein expression and increased metabolic activity (basal oxygen consumption, proton leak, and maximum respiration). By utilizing this “browning” process, the abundant and accessible WAT stem cell population can be engineered into 3D tissue constructs with the metabolic capacity of native BAT, ultimately for therapeutic intervention in vivoand as a tool for studying BAT and its metabolic properties.

The vast opportunities for biomaterials design and functionality enabled by mimicking nature continue to stretch the limits of imagination. As both biological understanding and engineering capabilities develop, more sophisticated biomedical materials can be synthesized that have multifaceted chemical, biological and physical characteristics designed to achieve specific therapeutic goals. Mimicry is being used in the design of polymers for biomedical applications that are required locally in tissues, systemically throughout the body, and at the interface with tissues.

Osteoarthritis (OA) is a degenerative disease associated with cartilage degradation, osteophyte formation, and fibrillation. Autologous Protein Solution (APS), a type of autologous anti-inflammatory orthobiologic, is used for pain management and treatment of OA. Various compositions of autologous PRP formulations are in clinical use for musculoskeletal pathologies, by nature of their minimal processing and source of bioactive molecules. Currently, there is no consensus on the optimal composition of the complex mixture. In this study, we focused on elucidating the immune cell subtypes and phenotypes in APS. We identified the immune cell types in APS from healthy donors and investigated phenotypic changes in the immune cells after APS processing. Based on flow cytometric analysis, we found that neutrophils and T cells are the most abundant immune cell types in APS, while monocytes experience the largest fold change in concentration compared to WBCs. Gene expression profiling revealed that APS processing results in differential gene expression changes dependent on immune cell type, with the most significantly differentially regulated genes occurring in the monocytes. Our results demonstrate that the mechanical processing of blood, whose main purpose is enrichment and separation, can alter its protein and cellular composition, as well as cellular phenotypes in the final product.

Tissue-engineered approaches to regenerate bone in the craniomaxillofacial region utilize biomaterial scaffolds to provide structural and biological cues to stem cells to stimulate osteogenic differentiation. Bioactive scaffolds are typically comprised of natural components but often lack the manufacturability of synthetic materials. To circumvent this trade-off, we 3D printed materials comprised of decellularized bone (DCB) matrix particles combined with polycaprolactone (PCL) to create novel hybrid DCB:PCL scaffolds for bone regeneration. Hybrid scaffolds were readily printable at compositions of up to 70% bone by mass and displayed robust mechanical properties. Assessments of surface features revealed both collagenous and mineral components of bone were present. Qualitative and quantitative assessments showed increased surface roughness relative to that of pure PCL scaffolds. These findings correlated with enhanced cell adhesion on hybrid surfaces relative to that on pure surfaces. Human adipose-derived stem cells (hASCs) cultured in

Tissue-engineering strategies offer promising tools for repairing cartilage damage; however, these strategies suffer from limitations under pathological conditions. As a model disease for these types of nonideal systems, the inflammatory environment in an osteoarthritic (OA) joint limits the efficacy of engineered therapeutics by disrupting joint homeostasis and reducing its capacity for regeneration. In this work, we investigated a sugar-based drug candidate, a tributanoylated N-acetyl-d-glucosamine analogue, called 3,4,6-O-Bu3GlcNAc, that is known to reduce nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) signaling in osteoarthritis. 3,4,6-O-Bu3GlcNAc not only inhibited NFκB signaling but also exerted chondrogenic and anti-inflammatory effects on chondrocytes isolated from patients with osteoarthritis. 3,4,6-O-Bu3GlcNAc also increased the expression of extracellular matrix proteins and induced cartilage tissue production in three-dimensional in vitrohydrogel culture systems. To translate these chondrogenic and anti-inflammatory properties to tissue regeneration in osteoarthritis, we implanted 3,4,6-O-Bu3GlcNAc-loaded poly(lactic-co-glycolic acid) microfiber scaffolds into rats. The drug-laden scaffolds were biocompatible, and when seeded with human OA chondrocytes, similarly promoted cartilage tissue formation. 3,4,6-O-Bu3GlcNAc combined with the appropriate structural environment could be a promising therapeutic approach for osteoarthritis.

Orthopedic tissue regeneration would benefit the aging population or patients with degenerative bone and cartilage diseases, especially osteoporosis and osteoarthritis. Despite progress in surgical and pharmacological interventions, new regenerative approaches are needed to meet the challenge of creating bone and articular cartilage tissues that are not only structurally sound but also functional, primarily to maintain mechanical integrity in their high load-bearing environments. In this review, we discuss new advances made in exploiting the three classes of materials in bone and cartilage regenerative medicine—cells, biomaterial-based scaffolds, and small molecules—and their successes and challenges reported in the clinic. In particular, the focus will be on the development of tissue-engineered bone and cartilage ex vivo by combining stem cells with biomaterials, providing appropriate structural, compositional, and mechanical cues to restore damaged tissue function. In addition, using small molecules to locally promote regeneration will be discussed, with potential approaches that combine bone and cartilage targeted therapeutics for the orthopedic-related disease, especially osteoporosis and osteoarthritis.

Aqueous tear-deficient dry eye disease is a multifactorial chronic disorder, in which the lacrimal gland fails to produce enough tears to maintain a healthy ocular surface. Some severe cases may develop corneal damage and significant vision loss. Treatment primarily involves palliation using ocular surface lubricants, but can only provide temporary relief. Construction of a bioengineered lacrimal gland having functional secretory epithelial cells is a potentially promising option for providing long-term relief to severe dry eye patients. Using sphere-forming culture techniques, we cultured adult rabbit lacrimal gland progenitor cells and prepared a lacrimal gland scaffold by decellularization. When progenitor cells were seeded onto the decellularized scaffold, they formed duct- and acinar-like structures in the three-dimensional culture system. Lacrimal gland epithelial cells showed good cell viability, cell differentiation, and secretory function in decellularized lacrimal gland matrix, as indicated by morphology, immunostaining, and β-hexosaminidase secretion assay. This study demonstrated the potential suitability of utilizing tissue-specific progenitor cells and a tissue-derived bioscaffold for lacrimal gland restoration.

Cell and protein arrays have demonstrated remarkable utility in the high-throughput evaluation of biological responses; however, they lack the complexity of native tissue and organs. Here we spotted tissue extracellular matrix (ECM) particles as two-dimensional (2D) arrays or incorporated them with cells to generate three-dimensional (3D) cell-matrix microtissue arrays. We then investigated the responses of human stem, cancer and immune cells to tissue ECM arrays originating from 11 different tissues. We validated the 2D and 3D arrays as representative of the in vivo microenvironment by means of quantitative analysis of tissue-specific cellular responses, including matrix production, adhesion and proliferation, and morphological changes after culture. The biological outputs correlated with tissue proteomics, and network analysis identified several proteins linked to cell function. Our methodology enables broad screening of ECMs to connect tissue-specific composition with biological activity, providing a new resource for biomaterials research and further understanding of regeneration and disease mechanisms.

IMPORTANCE: Acquired soft-tissue injury with resultant volume loss may cause significant deformity in size, shape, and body or facial contour. Current autologous fat transfer techniques have several limitations, including availability, donor site morbidity, and unpredictable rates of resorption. We present an extracellular matrix (ECM) biomaterial derived from human adipose tissue as an off-the-shelf alternative for soft-tissue volume restoration and compare clinically relevant rheologic properties. OBJECTIVES: To determine the rheologic properties of adipose-derived ECM and to compare it with lipoaspirate, calcium hydroxylapatite, and cross-linked hyaluronic acid. DESIGN, SETTING, AND PARTICIPANTS: Adipose-derived ECM (n = 4), lipoaspirate acquired from aesthetic liposuction (n = 4), calcium hydroxylapatite (n = 4), and cross-linked hyaluronic acid (n = 4) were obtained to determine the viscoelastic properties. INTERVENTIONS: Dynamic frequency oscillation measurements were conducted using a rheometer (ARES-G2; TA Instruments). All injections were performed using a 20-gauge needle, and all measurements were performed using serrated 25-mm parallel-plate geometry with a 1.0-mm gap at 37°C. MAIN OUTCOMES AND MEASURES: Oscillation measurements for storage modulus, a measure of the elastic properties, and complex viscosity were obtained over an angular frequency range of 0.01 to 100 rad/s. RESULTS: At 1 Hz, adipose-derived ECM had a mean (SD) storage modulus of 713.2 (42.9) Pa and a mean (SD) complex viscosity of 115.8 (6.9) Pa/s. Lipoaspirate had a mean (SD) storage modulus of 382.1 (66.8) Pa and a mean (SD) complex viscosity of 61.5 (10.7) Pa/s. Calcium hydroxylapatite had a mean (SD) storage modulus of 1122.1 (67.1) Pa and a mean (SD) complex viscosity of 207.2 (11.6) Pa/s. Cross-linked hyaluronic acid had a mean (SD) storage modulus of 63.9 (3.0) Pa and a mean (SD) complex viscosity of 10.9 (0.5) Pa/s. CONCLUSIONS AND RELEVANCE: Of the 4 materials tested, calcium hydroxylapatite has the highest mean storage modulus and mean complex viscosity, and hyaluronic acid has the lowest. The viscoelastic properties of adipose-derived ECM are most similar to those of lipoaspirate, suggesting that it may be an ideal candidate for soft-tissue reconstruction. LEVEL OF EVIDENCE: NA.

IMPORTANCE: Laryngotracheal stenosis (LTS) lacks an ideal animal model to study its unique wound-healing pathophysiology and the effect of interventions. OBJECTIVE: To present an in vivo, in situ mouse model of LTS that can be used to investigate its pathophysiology, mechanisms, and interventions for treatment. DESIGN, SETTING, AND SUBJECTS: Prospective controlled animal study performed at an academic animal research facility on 87 C57BL/6 mice. INTERVENTIONS: Experimental mice (n = 40) underwent bleomycin-coated wire-brush injury to the larynx and trachea, while mechanical injury controls (n = 32) underwent phosphate-buffered saline–coated wire-brush injury. Normal controls (n = 9) underwent no intervention, and mock surgery controls (n = 6) underwent anterior transcervical tracheal exposure only. Laryngotracheal complexes were harvested at days 7, 14, and 21 after injury. At the respective time points, mice in the chemomechanical and mechanical injury groups were killed, and their laryngotracheal complexes were harvested for histologic analysis. Normal and mock surgery controls were killed and then underwent histologic analysis. MAIN OUTCOMES AND MEASURES: The primary outcome measure was lamina propria thickness. RESULTS: The chemomechanical injury group maintained a significant increase in lamina propria thickness through day 21 compared with uninjured controls at day 7 (82.7 vs 41.8 μm; P&lt;.05), day 14 (93.5 vs 26.0 μm; P&lt;.05), and day 21 (91.2 vs 40.8 μm; P&lt;.05). Compared with the mechanical injury group, the chemomechanical injury group demonstrated a significantly increased thickness at 21 days (91.2 vs 33.7 μm; P&lt;.05). CONCLUSIONS AND RELEVANCE: Chemomechanical initiation of fibrosis in situ creates a viable mouse model of LTS that incorporates the physiologic circulatory supply and airflow. This small-animal model may be used to investigate the pathogenesis and inflammatory mechanisms of iatrogenic LTS and test therapeutic interventions to reverse or reduce the development of fibrosis.

This study was performed to evaluate the potential of a chondroitin sulfate-polyethylene glycol (CS-PEG) adhesive and collagen-based membrane (collagen vitrigel, CV) combination as a method to treat penetrating ocular injuries on the battlefield and to improve this method with two technologies: an antibiotic releasing CS-PEG adhesive and a corneal shaped CV. Burst testing using porcine cadaveric eyes, high-performance liquid chromatography, the Kirby-Bauer bacterial inhibition test, and CV implantations on the live and cadaveric rabbit eyes were performed. The ocular burst test showed CS-PEG adhesive could successfully repair 5-mm to 6-mm length wounds in the corneal and corneoscleral regions but would require CS-PEG + CV to treat larger wounds similar to those seen on the battlefield. In addition, high performance liquid chromatography and the Kirby-Bauer bacterial inhibition test presented evidence suggesting the vancomycin incorporated CS-PEG could inhibit Staphylococcus infection for 9 days. Furthermore, the curved CV showed an advantage by matching the corneal contour without any wrinkle formation. Although this pilot study showed a limited range of possible applications, we demonstrated that the combination of CS-PEG adhesive + CV is a promising method and the 2 technologies improve their applicability to the special demands of the battlefield.

IMPORTANCE Keloid lesions form because of alterations in the mechanisms that govern cutaneous wound healing. Although matrix metalloproteinases (MMPs) have been implicated in keloid pathophysiology, many questions still remain about their involvement. Our incomplete understanding of keloid pathophysiology has led to high recurrence rates in current treatments. No reliable animal model is available for studying keloids. OBJECTIVE To gain a better understanding of the disease mechanisms involved in keloid lesions in the hopes of identifying therapeutic options. DESIGN Fibroblasts derived from keloid tissue were incorporated in either Matrigel or polyethylene glycol diacrylate mixed with type I collagen to create 3-dimensional models to investigate the role MMPs play in keloid formation. The MMP gene expressions were also compared between fibroblasts isolated from different sites within the same keloid lesion. SETTING The Johns Hopkins School of Medicine, Baltimore, Maryland. PARTICIPANTS Keloid fibroblasts were received from the Baylor College of Medicine, and additional keloid fibroblasts were enzymatically isolated from the dermal layer of lesions removed from consenting patients at The Johns Hopkins Hospital. RESULTS In the Matrigel system, MMP9 and MMP13 were observed to be significantly upregulated in keloid fibroblasts. The addition of decorin resulted in a significant decrease of type I collagen and MMP1, MMP9, and MMP13 gene expressions from keloid fibroblasts. Higher MMP gene expressions were observed in fibroblasts isolated from the margins of the original keloid wound. CONCLUSIONS AND RELEVANCE MMP9 and MMP13 are expressed significantly more in keloid-derived cells, thus making them 2 potential targets for disease modification. Molecules that target organization of the lesion’s matrix can be beneficial in downregulating increased markers during the disease. In addition, heterogeneity is observed with the varying MMP gene expressions from site-specific fibroblasts within the same keloid lesion.

Cellular bioenergetics and redox (reduction–oxidation) play an important role in cell proliferation and differentiation, key aspects of building new tissues. In the present study, we examined the metabolic characteristics of human adipose-derived stem cells (hASCs) during proliferation and differentiation in both monolayer and three-dimensional biomaterial scaffolds. In monolayer, hASCs exhibited higher glycolysis and lower ox-phos as compared to both adipogenic and osteogenic differentiated cells, and hASCs demonstrated the Warburg effect (aerobic glycolysis). However, reactive oxygen species (ROS) levels increased during adipogenic differentiation, but decreased during osteogenic differentiation. Similarly, a decrease in ROS levels along with a higher mitochondrial membrane potential and viability was observed in hASCs encapsulated in poly(ethylene glycol) (PEG) hydrogels containing an adhesion peptide (RGD), compared to PEG hydrogels with a scrambled control peptide (GRD), demonstrating that adhesion-dependent signaling can also regulate ROS production and bioenergetics. As a result, we hypothesized that we could modulate osteogenesis in PEG hydrogels containing the adhesion peptide (RGD) by further reducing ROS levels using a small therapeutic molecule, L-carnitine, a metabolite with purported antioxidant effects. We observed reduced ROS levels, no effect on mitochondrial membrane potential, and increased osteogenic differentiation and tissue production in cells in the presence of L-carnitine. These results suggest the potential to manipulate tissue production by modulating cellular metabolism.

Inflammation and tissue degeneration play key roles in numerous rheumatic diseases, including osteoarthritis (OA). Efforts to reduce and effectively repair articular cartilage damage in an osteoarthritic environment are limited in their success due to the diseased environment. Treatment strategies focused on both reducing inflammation and increasing tissue production are necessary to effectively treat OA from a tissue-engineering perspective. In this work, we investigated the anti-inflammatory and tissue production capacity of a small molecule 3,4,6-O-tributanoylated-N-acetylglucosamine (3,4,6-O-Bu3GlcNAc) previously shown to inhibit the nuclear factor κB (NFκB) activity, a key transcription factor regulating inflammation. To mimic an inflammatory environment, chondrocytes were stimulated with interleukin-1β (IL-1β), a potent inflammatory cytokine. 3,4,6-O-Bu3GlcNAc exposure decreased the expression of NFκB target genes relevant to OA by IL-1β-stimulated chondrocytes after 24 h of exposure. The capacity of 3,4,6-O-Bu3GlcNAc to stimulate extracellular matrix (ECM) accumulation by IL-1β-stimulated chondrocytes was evaluated in vitroutilizing a three-dimensional hydrogel culturing system. After 21 days, 3,4,6-O-Bu3GlcNAc exposure induced quantifiable increases in both sulfated glycosaminoglycan and total collagen. Histological staining for proteoglycans and type II collagen confirmed these findings. The increased ECM accumulation was not due to the hydrolysis products of the small molecule, n-butyrate and N-acetylglucosamine (GlcNAc), as the isomeric 1,3,4-O-tributanoylated N-acetylglucosamine (1,3,4-O-Bu3GlcNAc) did not elicit a similar response. These findings demonstrate that a novel butanoylated GlcNAc derivative, 3,4,6-O-Bu3GlcNAc, has the potential to stimulate new tissue production and reduce inflammation in IL-1β-induced chondrocytes with utility for OA and other forms of inflammatory arthritis.

The weak intrinsic meniscus healing response and technical challenges associated with meniscus repair contribute to a high rate of repair failures and meniscectomies. Given this limited healing response, the development of biologically active adjuncts to meniscal repair may hold the key to improving meniscal repair success rates. This study demonstrates the development of a bone marrow (BM) adhesive that binds, stabilizes, and stimulates fusion at the interface of meniscus tissues. Hydrogels containing several chondroitin sulfate (CS) adhesive levels (30, 50, and 70 mg/mL) and BM levels (30%, 50%, and 70%) were formed to investigate the effects of these components on hydrogel mechanics, bovine meniscal fibrochondrocyte viability, proliferation, matrix production, and migration ability in vitro. The BM content positively and significantly affected fibrochondrocyte viability, proliferation, and migration, while the CS content positively and significantly affected adhesive strength (ranged from 60±17 kPa to 335±88 kPa) and matrix production. Selected material formulations were translated to a subcutaneous model of meniscal fusion using adhered bovine meniscus explants implanted in athymic rats and evaluated over a 3-month time course. Fusion of adhered meniscus occurred in only the material containing the highest BM content. The technology can serve to mechanically stabilize the tissue repair interface and stimulate tissue regeneration across the injury site.

Spontaneous differentiation of human embryonic stem cells (hESCs) is generally inefficient and leads to a heterogeneous population of differentiated and undifferentiated cells, limiting the potential use of hESCs for cell-based therapy and studies of specific differentiation programs. Here, we demonstrate biomaterial-dependent commitment of a mesenchymal cell population derived from hESCs toward the osteogenic lineage in vivo. In skeletal development, bone formation from condensing mesenchymal cells involves two distinct pathways: endochondral and intramembraneous bone formation. In this study, we demonstrate that the hESC-derived mesenchymal cells differentiate and regenerate in vivobone tissues through two different pathways depending upon the local cues present in a scaffold microenvironment. Hydroxyapatite (HA) was incorporated into biodegradable poly(lactic-co-glycolic acid)/poly(l-lactic acid) (PLGA/PLLA) scaffolds to enhance bone formation. The HA microenvironment stabilized the β-catenin and upregulated Runx2, resulting in faster bone formation through intramembraneous ossification. hESC-derived mesenchymal cells seeded on the PLGA/PLLA scaffold without HA, however, showed minimal levels Runx2, and differentiated via endochondral ossification, as evidenced by formation of cartilaginous tissue, followed by calcification and increased blood vessel invasion. These results indicate that the ossification mechanisms of the hESC-derived mesenchymal stem cells can be regulated by the scaffold-mediated microenvironments, and bone tissue can be formed.

Hyaluronic acid (HA) is an extracellular matrix molecule with multiple physical and biological functions found in many tissues, including cartilage. HA has been incorporated in a number of biomaterial and scaffold systems. However, HA in the material may be difficult to control if it is not chemically modified and chemical modification of HA may negatively impact biological function. In this study, we developed a poly(ethylene glycol) hydrogel with noncovalent HA-binding capabilities and evaluated its ability to support cartilage formation in vitroand in an articular defect model. Chondrogenic differentiation of mesenchymal stem cells encapsulated in the HA-interactive scaffolds containing various amounts of exogenous HA was evaluated. The HA-binding hydrogel without exogenous HA produced the best cartilage as determined by biochemical content (glysocaminoglycan and collagen), histology (Safranin O and type II collagen staining), and gene expression analysis for aggrecan, type I collagen, type II collagen,and sox-9. This HA-binding formulation was then translated to an osteochondral defect model in the rat knee. After 6 weeks, histological analysis demonstrated improved cartilage tissue production in defects treated with the HA-interactive hydrogel compared to noninteractive control scaffolds and untreated defects. In addition to the tissue repair in the defect space, the Safranin O staining in cartilage tissue surrounding the defect was greater in treatment groups where the HA-binding scaffold was applied. In sum, incorporation of a noncovalent HA-binding functionality into biomaterials provides an ability to interact with local or exogenous HA, which can then impact tissue remodeling and ultimately new tissue production.

Electrospun fibers based on aliphatic polyesters, such as poly(ε-caprolactone) (PCL), have been widely used in regenerative medicine and drug delivery applications due to their biocompatibility, low cost and ease of fabrication. However, these aliphatic polyester fibers are hydrophobic in nature, resulting in poor wettability, and they lack functional groups for decorating the scaffold with chemical and biological cues. Current strategies employed to overcome these challenges include coating and blending the fibers with bioactive components or chemically modifying the fibers with plasma treatment and reactants. In the present study, we report on designing multifunctional electrospun nanofibers based on the inclusion complex of PCL-α-cyclodextrin (PCL-α-CD), which provides both structural support and multiple functionalities for further conjugation of bioactive components. This strategy is independent of any chemical modification of the PCL main chain, and electrospinning of PCL-α-CD is as easy as electrospinning PCL. Here, we describe synthesis of the PCL-α-CD electrospun nanofibers, elucidate composition and structure, and demonstrate the utility of functional groups on the fibers by conjugating a fluorescent small molecule and a polymeric-nanobead to the nanofibers. Furthermore, we demonstrate the application of PCL-α-CD nanofibers for promoting osteogenic differentiation of human adipose-derived stem cells (hADSCs), which induced a higher level of expression of osteogenic markers and enhanced production of extracellular matrix (ECM) proteins or molecules compared with control PCL fibers.

Soft-tissue repair is currently limited by the availability of autologous tissue sources and the absence of an ideal soft-tissue replacement comparable to native adipose tissue. Extracellular matrix–based biomaterials have demonstrated great potential as instructive scaffolds for regenerative medicine, mechanically and biochemically defined by the tissue of origin. As such, the distinctive high lipid content of adipose tissue requires unique processing conditions to generate a biocompatible scaffold for soft-tissue repair.

Objective: To characterize and evaluate osteoarthritic (OA) chondrocytes, in comparison to normal chondrocytes, through a novel 3-dimensional (3-D) culture system, poly(ethylene-glycol) diacrylate (PEGDA). The cytokine interleukin 1β (IL-1β) was also used to simulate an in vitroOA model.Methods: Normal and OA chondrocytes were cultured in monolayer and analyzed for changes in cartilage-specific gene expressions due to passage number. Then, cells were encapsulated in PEGDA to evaluate phenotype and matrix production capabilities through the in vitroculture system. Characterization was conducted with polymerase chain reaction (PCR), biochemical analyses, and histological staining. 3-D encapsulated chondrocytes (human and bovine) were also treated with IL-1β to characterize how the cytokine affects gene transcription and extracellular matrix (ECM) content.Results: In 2-dimensional monolayer, anabolic genes were down-regulated significantly in both normal and OA chondrocytes. In 3-D culture, OA chondrocytes demonstrated significantly higher expressions of catabolic genes when compared to normal cells. Differentiation medium resulted in significantly more matrix production than growth medium from OA chondrocytes, indicated through histological staining. In addition, normal chondrocytes responded more significantly to exogenous administration of IL-1β than OA chondrocytes. Temporary initial stimulation of IL-1β to OA chondrocytes resulted in comparable gene expressions to untreated cells after 3 weeks of in vitroculture.Conclusions: Our findings demonstrate the use of OA chondrocytes in tissue engineering and their significance for potential future cartilage regeneration research through their matrix production capabilities and the use of a hydrogel culture system.

Laboratory investigations of stems cells in regenerative medicine have generated considerable interest within recent years, however some of this excitement is yet to be matched in the clinical arena. Two fields that are well poised to make significant clinical impact in the coming years are those of cartilage and corneal regeneration. In the case of cornea, it is widely acknowledged that corneal epithelium is derived from an adult stem cell type resident within the cornea. These cells, known as limbal stem cells (LSCs), have been widely investigated for their ex-vivo culture and subsequent transplantation efficacy, with some techniques already enjoying limited clinical application. Thus far however, only preliminary evidence currently exists to suggest that there is a population of adult stem cells which gives rise to stromal keratocytes or to the corneal endothelium. A handful of reports have discussed studies in which non-LSC adult stem cells such as mesenchymal stems cells (MSCs) or embryonic stem cells (ESCs) are being applied to corneal regeneration. Though adult stem cells have been shown to exist in articular cartilage, they have proven elusive, which corroborates the limited ability of this tissue to self-repair. Rather, MSCs, ESCs as well as adipose-derived, periosteum-derived, muscle-derived and synovium-derived stem cells (ADCs, PDCs, MDCs and SDCs respectively) are being extensively explored for cartilage regeneration. This review discusses emerging trends in the applications of both adult and embryonic stem cells to cartilage and corneal regeneration, with an emphasis on those techniques that have been applied clinically or which show significant potential for clinical translation.

There is an extensive clinical need for soft tissue filler materials, such as adipose tissue, for plastic and reconstructive surgery. Due to limitations with autologous adipose transplantation, engineered adipose tissue provides a potential alternative therapy. Embryonic germ cells form embryoid bodies and subsequent embryoid body–derived (EBD) cells have the ability to differentiate toward multiple tissue types. The objective of this study was to demonstrate that EBD cells were capable of adipogenic differentiation in vitroand in vivousing a poly(ethylene glycol)-based hydrogel scaffold. EBD cells underwent adipogenic differentiation in vitroand in vivo. Results were directly compared to adipogenic differentiation of adult bone marrow–derived mesenchymal stem cells (MSCs). Differentiated EBD cells in both monolayer and three-dimensional in vitroculture demonstrated fat granules by light microscopy, stained positive for lipids with oil red-O, and expressed adipocyte-specific genes (lipoprotein lipase [LPL], peroxisome proliferator activated receptor γ2, and adipocyte-specific fatty acid binding protein [αP2]). In vivoconstructs demonstrated adipogenic differentiation by αP2 and LPL gene expression and oil red-O staining of lipid granules. In conclusion, EBD cells are capable of differentiating toward an adipogenic lineage in vitroand in vivo. EBD cells' adipogenic differentiation is comparable to that of MSCs and demonstrate therapeutic potential for soft tissue augmentation and reconstruction.

Emerging insights into cellular senescence highlight the relevance of senescence in musculoskeletal disorders, which represent the leading global cause of disability. Cellular senescence was initially described by Hayflick et al. in 1961 as an irreversible nondividing state in in vitro cell culture studies. We now know that cellular senescence can occur in vivo in response to various stressors as a heterogeneous and tissue-specific cell state with a secretome phenotype acquired after the initial growth arrest. In the past two decades, compelling evidence from preclinical models and human data show an accumulation of senescent cells in many components of the musculoskeletal system. Cellular senescence is therefore a defining feature of age-related musculoskeletal disorders, and targeted elimination of these cells has emerged recently as a promising therapeutic approach to ameliorate tissue damage and promote repair and regeneration of the skeleton and skeletal muscles. In this review, we summarize evidence of the role of senescent cells in the maintenance of bone homeostasis during childhood and their contribution to the pathogenesis of chronic musculoskeletal disorders, including osteoporosis, osteoarthritis, and sarcopenia. We highlight the diversity of the senescent cells in the microenvironment of bone, joint, and skeletal muscle tissue, as well as the mechanisms by which these senescent cells are involved in musculoskeletal diseases. In addition, we discuss how identifying and targeting senescent cells might positively affect pathologic progression and musculoskeletal system regeneration.

Restoration of soft tissue defects relies on proper structural support and bioactive components to instruct remodeling processes that have tightly controlled spatial and temporal activity not yet fully understood. One clinical mainstay for soft tissue reconstruction, autologous fat grafting, has widespread use in reconstructive and esthetic surgeries owing to the success of liposuction techniques in the 1980s and standardization of adipose processing in the 1990s. Although widespread, clinical outcomes are unpredictable owing to significant variation in fat resorption rates (25–80%) [1–4] and a lack of assays to predict outcomes or confirm cell viability preoperatively. Autologous fat grafting has a few limitations that have fueled the development of off-the-shelf technologies using tissue engineering approaches. Acellular human tissue products have an opportunity to meet this significant clinical unmet need. Selected recent strategies in acellular adipose tissue products for soft tissue reconstruction will be reviewed here. These advancements will inform further development for soft tissue reconstruction.

A new type of synthetic hydrogel scaffold that mimics certain aspects of structure and function of natural extracellular matrix (ECM) has been developed. We previously reported the conjugation of collagen mimetic peptide (CMP) to poly(ethylene oxide) diacrylate (PEODA) to create a polymer-peptide hybrid scaffold for a suitable cell microenvironment. In this study, we showed that the CMP-mediated microenvironment enhances the chondrogenic differentiation of mesenchymal stem cells (MSCs). MSCs were harvested and photo-encapsulated in CMP-conjugated PEODA (CMP/PEODA). After 3 weeks, the histological and biochemical analysis of the CMP/PEODA gel revealed twice as much glycosaminoglycan and collagen contents as in control PEODA hydrogels. Moreover, MSCs cultured in CMP/PEODA hydrogel exhibited a lower level of hypertrophic markers, core binding factor alpha 1, and type X collagen than MSCs in PEODA hydrogel as revealed by gene expression and immunohistochemisty. These results indicate that CMP/PEODA hydrogel provides a favorable microenvironment for encapsulated MSCs and regulates their downstream chondrogenic differentiation.

Surgical options for cartilage reconstruction can be significantly improved through advances in cartilage tissue engineering, whereby functional tissue replacements are created by growing cells on polymer scaffolds. The objective of this study was to use a photopolymerizable hydrogel to implant bone marrow–derived mesenchymal stem cells subcutaneously in a minimally invasive manner and promote cartilage tissue formation by the cells in vivo.

This article summarizes the views expressed at the third session of the workshop "Tissue Engineering—The Next Generation," which was devoted to the engineering of complex tissue structures. Antonios Mikos described the engineering of complex oral and craniofacial tissues as a "guided interplay" between biomaterial scaffolds, growth factors, and local cell populations toward the restoration of the original architecture and function of complex tissues. Susan Herring, reviewing osteogenesis and vasculogenesis, explained that the vascular arrangement precedes and dictates the architecture of the new bone, and proposed that engineering of osseous tissues might benefit from preconstruction of an appropriate vasculature. Jennifer Elisseeff explored the formation of complex tissue structures based on the example of stratified cartilage engineered using stem cells and hydrogels. Helen Lu discussed engineering of tissue interfaces, a problem critical for biological fixation of tendons and ligaments, and the development of a new generation of fixation devices. Rita Kandel discussed the challenges related to the re-creation of the cartilage-bone interface, in the context of tissue engineered joint repair. Frederick Schoen emphasized, in the context of heart valve engineering, the need for including the requirements derived from "adult biology" of tissue remodeling and establishing reliable early predictors of success or failure of tissue engineered implants. Mehmet Toner presented a review of biopreservation techniques and stressed that a new breakthrough in this field may be necessary to meet all the needs of tissue engineering. David Mooney described systems providing temporal and spatial regulation of growth factor availability, which may find utility in virtually all tissue engineering and regeneration applications, including directed in vitroand in vivovascularization of tissues. Anthony Atala offered a clinician's perspective for functional tissue regeneration, and discussed new biomaterials that can be used to develop new regenerative technologies.

Human embryonic stem cells (hESCs) have the potential to self-renew and generate multiple cell types, producing critical building blocks for tissue engineering and regenerative medicine applications. Here, we describe the efficient derivation and chondrogenic differentiation of mesenchymal-like cells from hESCs. These cells exhibit mesenchymal stem cell (MSC) surface markers, including CD29, CD44, CD105, and platelet-derived growth factor receptor-α. Under appropriate growth conditions, the hESC-derived cells proliferated without phenotypic changes and maintained MSC surface markers. The chondrogenic capacity of the cells was studied in pellet culture and after encapsulation in poly(ethylene glycol)-diacrylate (PEGDA) hydrogels with exogenous extracellular proteins or arginineglycine- aspartate (RGD)-modified PEGDA hydrogels. The hESC-derived cells exhibited growth factor– dependent matrix production in pellet culture but did not produce tissue characteristic of cartilage morphology. In PEGDA hydrogels containing exogenous hyaluronic acid or type I collagen, no significant cell growth or matrix production was observed. In contrast, when these cells were encapsulated in RGDmodified poly(ethylene glycol)hydrogels, neocartilage with basophilic extracellular matrix deposition was observed within 3 weeks of culture, producing cartilage-specific gene up-regulation and extracellular matrix production. Our results indicate that precursor cells characteristic of a MSC population can be cultured from differentiating hESCs through embryoid bodies, thus holding great promise for a potentially unlimited source of cells for cartilage tissue engineering.

Bioresponsive and intelligent biomaterials are a vehicle for manipulating cell function to promote tissue development and/or tissue engineering. A photopolymerized hydrogel based on a phosphoester– poly(ethylene glycol) polymer (PhosPEG) was synthesized for application to marrow-derived mesenchymal stem cell (MSC) encapsulation and tissue engineering of bone. The phosphor-containing hydrogels were hydrolytically degradable and the rate of degradation increased in the presence of a bone-derived enzyme, alkaline phosphatase. Gene expression and protein analysis of encapsulated MSCs demonstrated that PhosPEG–PEG cogels containing an intermediate concentration of phosphorus promoted the gene expression of bone-specific markers including type I collagen, alkaline phosphatase, and osteonectin, without the addition of growth factors or other biological agents, compared with pure poly(ethylene glycol)-based gels. Secretion of alkaline phosphatase, osteocalcin, and osteonectin protein was also increased in the PhosPEG cogels. Mineralization of gels increased in the presence of phosphorus in both cellular and acellular constructs compared with PEG gels. In summary, phosphate-PEG-derived hydrogels increase gene expression of bone-specific markers, secretion of bone-related matrix, and mineralization and may have a potential impact on bone-engineering therapies.

Cartilage is the tissue that lines the surface of bones in articulating joints, allowing painless joint movement. Cartilage loss is an increasingly significant problem, particularly with the ageing of active baby boomers, with few efficacious treatments available at present. Tissue engineering is a field that has evolved over recent years to combat tissue loss by providing a living tissue equivalent or substitute that can mimic the properties of the lost tissue. The general strategy of tissue engineering is to place cells on a biomaterial scaffold that is designed to promote cell function and form new tissue. This review describes the status of materials that are available as injectable scaffolds for tissue engineering and the numerous cell types that can be applied to cartilage repair, including cells derived from cartilage and stem cells. The current state of injectable cartilage tissue engineering and the hurdles that remain for widespread clinical application are discussed.

Uniform design of synovial articulations across mammalian species is challenged by their common susceptibility to joint degeneration. The present study was designed to investigate the possibility of creating human-shaped articular condyles by rat bone marrow-derived mesenchymal stem cells (MSCs) encapsulated in a biocompatible poly(ethylene glycol)-based hydrogel. Rat MSCs were harvested, expanded in culture, and treated with either chondrogenic or osteogenic supplements. Rat MSC-derived chondrogenic and osteogenic cells were loaded in hydrogel suspensions in two stratified and yet integrated hydrogel layers that were sequentially photopolymerized in a human condylar mold. Harvested articular condyles from 4-week in vivoimplantation demonstrated stratified layers of chondrogenesis and osteogenesis. Parallel in vitroexperiments using goat and rat MSCs corroborated in vivodata by demonstrating the expression of chondrogenic and osteogenic markers by biochemical and mRNA analyses. Ex vivoincubated goat MSC-derived chondral constructs contained cartilage-related glycosaminoglycans and collagen. By contrast, goat MSC-derived osteogenic constructs expressed alkaline phosphatase and osteonectin genes, and showed escalating calcium content over time. Rat MSC-derived osteogenic constructs were stiffer than rat MSC-derived chondrogenic constructs upon nanoindentation with atomic force microscopy. These findings may serve as a primitive proof of concept for ultimate tissue-engineered replacement of degenerated articular condyles via a single population of adult mesenchymal stem cells.