Engineering Innervated Musculoskeletal Tissues for Regenerative Orthopedics and Disease Modeling.

Musculoskeletal (MSK) disorders significantly burden patients and society, resulting in high healthcare costs and productivity loss. These disorders are the leading cause of physical disability, and their prevalence is expected to increase as sedentary lifestyles become common and the global population of the elderly increases. Proper innervation is critical to maintaining MSK function, and nerve damage or dysfunction underlies various MSK disorders, underscoring the potential of restoring nerve function in MSK disorder treatment. However, most MSK tissue engineering strategies have overlooked the significance of innervation. This review first expounds upon innervation in the MSK system and its importance in maintaining MSK homeostasis and functions. This will be followed by strategies for engineering MSK tissues that induce post-implantation in situ innervation or are pre-innervated. Subsequently, research progress in modeling MSK disorders using innervated MSK organoids and organs-on-chips (OoCs) is analyzed. Finally, the future development of engineering innervated MSK tissues to treat MSK disorders and recapitulate disease mechanisms is discussed. This review provides valuable insights into the underlying principles, engineering methods, and applications of innervated MSK tissues, paving the way for the development of targeted, efficacious therapies for various MSK conditions.

Effect of Aging on Tendon Biology, Biomechanics and Implications for Treatment Approaches.

Tendon aging is associated with an increasing prevalence of tendon injuries and/or chronic tendon diseases, such as tendinopathy, which affects approximately 25% of the adult population. Aged tendons are often characterized by a reduction in the number and functionality of tendon stem/progenitor cells (TSPCs), fragmented or disorganized collagen bundles, and an increased deposition of glycosaminoglycans (GAGs), leading to pain, inflammation, and impaired mobility. Although the exact pathology is unknown, overuse and microtrauma from aging are thought to be major causative factors. Due to the hypovascular and hypocellular nature of the tendon microenvironment, healing of aged tendons and related injuries is difficult using current pain/inflammation and surgical management techniques. Therefore, there is a need for novel therapies, specifically cellular therapy such as cell rejuvenation, due to the decreased regenerative capacity during aging. To augment the therapeutic strategies for treating tendon-aging-associated diseases and injuries, a comprehensive understanding of tendon aging pathology is needed. This review summarizes age-related tendon changes, including cell behaviors, extracellular matrix (ECM) composition, biomechanical properties and healing capacity. Additionally, the impact of conventional treatments (diet, exercise, and surgery) is discussed, and recent advanced strategies (cell rejuvenation) are highlighted to address aged tendon healing. This review underscores the molecular and cellular linkages between aged tendon biomechanical properties and the healing response, and provides an overview of current and novel strategies for treating aged tendons. Understanding the underlying rationale for future basic and translational studies of tendon aging is crucial to the development of advanced therapeutics for tendon regeneration.

Enhancing the potential of aged human articular chondrocytes for high-quality cartilage regeneration.

Autologous chondrocyte implantation (ACI) is a regenerative procedure used to treat focal articular cartilage defects in knee joints. However, age has been considered as a limiting factor and ACI is not recommended for patients older than 40-50 years of age. One reason for this may be due to the reduced capacity of aged chondrocytes in generating new cartilage. Currently, the underlying mechanism contributing to aging-associated functional decline in chondrocytes is not clear and no proven approach exists to reverse chondrocyte aging. Given that chondrocytes in healthy hyaline cartilage typically display a spherical shape, believed to be essential for chondrocyte phenotype stability, we hypothesize that maintaining aged chondrocytes in a suspension culture that forces the cells to adopt a round morphology may help to "rejuvenate" them to a younger state, thus, leading to enhanced cartilage regeneration. Chondrocytes isolated from aged donors displayed reduced proliferation potential and impaired capacity in generating hyaline cartilage, compared to cells isolated from young donors, indicated by increased hypertrophy and cellular senescence. To test our hypothesis, the "old" chondrocytes were seeded as a suspension onto an agarose-based substratum, where they maintained a round morphology. After the 3-day suspension culture, aged chondrocytes displayed enhanced replicative capacity, compared to those grown adherent to tissue culture plastic. Moreover, chondrocytes subjected to suspension culture formed new cartilage in vitro with higher quality and quantity, with enhanced cartilage matrix deposition, concomitant with lower levels of hypertrophy and cellular senescence markers. Mechanistic analysis suggested the involvement of the RhoA and ERK1/2 signaling pathways in the "rejuvenation" process. In summary, our study presents a robust and straightforward method to enhance the function of aged human chondrocytes, which can be conveniently used to generate a large number of high-quality chondrocytes for ACI application in the elderly.

A High-Throughput Mechanical Activator for Cartilage Engineering Enables Rapid Screening of in vitro Response of Tissue Models to Physiological and Supra-Physiological Loads.

Articular cartilage is crucially influenced by loading during development, health, and disease. However, our knowledge of the mechanical conditions that promote engineered cartilage maturation or tissue repair is still incomplete. Current in vitro models that allow precise control of the local mechanical environment have been dramatically limited by very low throughput, usually just a few specimens per experiment. To overcome this constraint, we have developed a new device for the high throughput compressive loading of tissue constructs: the High Throughput Mechanical Activator for Cartilage Engineering (HiT-MACE), which allows the mechanoactivation of 6 times more samples than current technologies. With HiT-MACE we were able to apply cyclic loads in the physiological (e.g., equivalent to walking and normal daily activity) and supra-physiological range (e.g., injurious impacts or extensive overloading) to up to 24 samples in one single run. In this report, we compared the early response of cartilage to physiological and supra-physiological mechanical loading to the response to IL-1ß exposure, a common but rudimentary in vitro model of cartilage osteoarthritis. Physiological loading rapidly upregulated gene expression of anabolic markers along the TGF-ß1 pathway. Notably, TGF-ß1 or serum was not included in the medium. Supra-physiological loading caused a mild catabolic response while IL-1ß exposure drove a rapid anabolic shift. This aligns well with recent findings suggesting that overloading is a more realistic and biomimetic model of cartilage degeneration. Taken together, these findings showed that the application of HiT-MACE allowed the use of larger number of samples to generate higher volume of data to effectively explore cartilage mechanobiology, which will enable the design of more effective repair and rehabilitation strategies for degenerative cartilage pathologies.

Wdpcp regulates cellular proliferation and differentiation in the developing limb via hedgehog signaling.

BACKGROUND: Mice with a loss of function mutation in Wdpcp were described previously to display severe birth defects in the developing heart, neural tube, and limb buds. Further characterization of the skeletal phenotype of Wdpcp null mice was limited by perinatal lethality. RESULTS: We utilized Prx1-Cre mice to generate limb bud mesenchyme specific deletion of Wdpcp. These mice recapitulated the appendicular skeletal phenotype of the Wdpcp null mice including polydactyl and limb bud signaling defects. Examination of late stages of limb development demonstrated decreased size of cartilage anlagen, delayed calcification, and abnormal growth plates. Utilizing in vitro assays, we demonstrated that loss of Wdpcp in skeletal progenitors lead to loss of hedgehog signaling responsiveness and associated proliferative response. In vitro chondrogenesis assays showed this loss of hedgehog and proliferative response was associated with decreased expression of early chondrogenic marker N-Cadherin. E14.5 forelimbs demonstrated delayed ossification and expression of osteoblast markers Runx2 and Sp7. P0 growth plates demonstrated loss of hedgehog signaling markers and expansion of the hypertrophic zones of the growth plate. In vitro osteogenesis assays demonstrated decreased osteogenic differentiation of Wdpcp null mesenchymal progenitors in response to hedgehog stimulation. CONCLUSIONS: These findings demonstrate how Wdpcp and associated regulation of the hedgehog signaling pathway plays an important role at multiple stages of skeletal development. Wdpcp is necessary for positive regulation of hedgehog signaling and associated proliferation is key to the initiation of chondrogenesis. At later stages, Wdpcp facilitates the robust hedgehog response necessary for chondrocyte hypertrophy and osteogenic differentiation.

Tendon-derived extracellular matrix induces mesenchymal stem cell tenogenesis via an integrin/transforming growth factor-ß crosstalk-mediated mechanism.

Treatment of tendon injuries is challenging. To develop means to augment tendon regeneration, we have previously prepared a soluble, low immunogenic (DNA-free), tendon extracellular matrix fraction (tECM) by urea extraction of juvenile bovine tendons, which is capable of enhancing transforming growth factor-ß (TGF-ß) mediated tenogenesis in human adipose-derived stem cells (hASCs). Here, we aimed to elucidate the mechanism of tECM-driven hASC tenogenic differentiation in vitro, focusing on the integrin and TGF-ß/SMAD pathways. Our results showed that tECM promoted hASC proliferation and tenogenic differentiation in vitro based on tenogenesis-associated markers. tECM also induced higher expression of several integrin subunits and TGF-ß receptors, and nuclear translocation of p-SMAD2 in hASCs. Pharmacological inhibition of integrin-ECM binding, focal adhesion kinase (FAK) signaling, or TGF-ß signaling independently led to compromised pro-tenogenic effects of tECM and actin fiber polymerization. Additionally, integrin blockade inhibited tECM-driven TGFBR2 expression, while inhibiting TGF-ß signaling decreased tECM-mediated expression of integrin α1, α2, and ß1 in hASCs. Together, these findings suggest that the strong pro-tenogenic bioactivity of tECM is regulated via integrin/TGF-ß signaling crosstalk. Understanding how integrins interact with signaling by TGF-ß and/or other growth factors (GFs) within the tendon ECM microenvironment will provide a rational basis for an ECM-based approach for tendon repair.

Tissue Engineering for Musculoskeletal Regeneration and Disease Modeling.

Musculoskeletal injuries and associated conditions are the leading cause of physical disability worldwide. The concept of tissue engineering has opened up novel approaches to repair musculoskeletal defects in a fast and/or efficient manner. Biomaterials, cells, and signaling molecules constitute the tissue engineering triad. In the past 40 years, significant progress has been made in developing and optimizing all three components, but only a very limited number of technologies have been successfully translated into clinical applications. A major limiting factor of this barrier to translation is the insufficiency of two-dimensional cell cultures and traditional animal models in informing the safety and efficacy of in-human applications. In recent years, microphysiological systems, often referred to as organ or tissue chips, generated according to tissue engineering principles, have been proposed as the next-generation drug testing models. This chapter aims to first review the current tissue engineering-based approaches that are being applied to fabricate and develop the individual critical elements involved in musculoskeletal organ/tissue chips. We next highlight the general strategy of generating musculoskeletal tissue chips and their potential in future regenerative medicine research. Exemplary microphysiological systems mimicking musculoskeletal tissues are described. With sufficient physiological accuracy and relevance, the human cell-derived, three-dimensional, multi-tissue systems have been used to model a number of orthopedic disorders and to test new treatments. We anticipate that the novel emerging tissue chip technology will continually reshape and improve our understanding of human musculoskeletal pathophysiology, ultimately accelerating the development of advanced pharmaceutics and regenerative therapies.

Differences in neural stem cell identity and differentiation capacity drive divergent regenerative outcomes in lizards and salamanders.

While lizards and salamanders both exhibit the ability to regenerate amputated tails, the outcomes achieved by each are markedly different. Salamanders, such as Ambystoma mexicanum, regenerate nearly identical copies of original tails. Regenerated lizard tails, however, exhibit important morphological differences compared with originals. Some of these differences concern dorsoventral patterning of regenerated skeletal and spinal cord tissues; regenerated salamander tail tissues exhibit dorsoventral patterning, while regrown lizard tissues do not. Additionally, regenerated lizard tails lack characteristically roof plate-associated structures, such as dorsal root ganglia. We hypothesized that differences in neural stem cells (NSCs) found in the ependyma of regenerated spinal cords account for these divergent regenerative outcomes. Through a combination of immunofluorescent staining, RT-PCR, hedgehog regulation, and transcriptome analysis, we analyzed NSC-dependent tail regeneration. Both salamander and lizard Sox2+ NSCs form neurospheres in culture. While salamander neurospheres exhibit default roof plate identity, lizard neurospheres exhibit default floor plate. Hedgehog signaling regulates dorsalization/ventralization of salamander, but not lizard, NSCs. Examination of NSC differentiation potential in vitro showed that salamander NSCs are capable of neural differentiation into multiple lineages, whereas lizard NSCs are not, which was confirmed by in vivo spinal cord transplantations. Finally, salamander NSCs xenogeneically transplanted into regenerating lizard tail spinal cords were influenced by native lizard NSC hedgehog signals, which favored salamander NSC floor plate differentiation. These findings suggest that NSCs in regenerated lizard and salamander spinal cords are distinct cell populations, and these differences contribute to the vastly different outcomes observed in tail regeneration.

Macrophages Modulate the Function of MSC- and iPSC-Derived Fibroblasts in the Presence of Polyethylene Particles.

Fibroblasts in the synovial membrane secrete molecules essential to forming the extracellular matrix (ECM) and supporting joint homeostasis. While evidence suggests that fibroblasts contribute to the response to joint injury, the outcomes appear to be patient-specific and dependent on interactions between resident immune cells, particularly macrophages (Mφs). On the other hand, the response of Mφs to injury depends on their functional phenotype. The goal of these studies was to further explore these issues in an in vitro 3D microtissue model that simulates a pathophysiological disease-specific microenvironment. Two sources of fibroblasts were used to assess patient-specific influences: mesenchymal stem cell (MSC)- and induced pluripotent stem cell (iPSC)-derived fibroblasts. These were co-cultured with either M1 or M2 Mφs, and the cultures were challenged with polyethylene particles coated with lipopolysaccharide (cPE) to model wear debris generated from total joint arthroplasties. Our results indicated that the fibroblast response to cPE was dependent on the source of the fibroblasts and the presence of M1 or M2 Mφs: the fibroblast response as measured by gene expression changes was amplified by the presence of M2 Mφs. These results demonstrate that the immune system modulates the function of fibroblasts; furthermore, different sources of differentiated fibroblasts may lead to divergent results. Overall, our research suggests that M2 Mφs may be a critical target for the clinical treatment of cPE induced fibrosis.

Mechanism of traumatic heterotopic ossification: In search of injury-induced osteogenic factors.

Heterotopic ossification (HO) is a pathological condition of abnormal bone formation in soft tissue. Three factors have been proposed as required to induce HO: (a) osteogenic precursor cells, (b) osteoinductive agents and (c) an osteoconductive environment. Since Urist's landmark discovery of bone induction in skeletal muscle tissue by demineralized bone matrix, it is generally believed that skeletal muscle itself is a conductive environment for osteogenesis and that resident progenitor cells in skeletal muscle are capable of differentiating into osteoblast to form bone. However, little is known about the naturally occurring osteoinductive agents that triggered this osteogenic response in the first place. This article provides a review of the emerging findings regarding distinct types of HO to summarize the current understanding of HO mechanisms, with special attention to the osteogenic factors that are induced following injury. Specifically, we hypothesize that muscle injury-induced up-regulation of local bone morphogenetic protein-7 (BMP-7) level, combined with glucocorticoid excess-induced down-regulation of circulating transforming growth factor-ß1 (TGF-ß1) level, could be an important causative mechanism of traumatic HO formation.

Decellularized bone extracellular matrix in skeletal tissue engineering.

Bone possesses an intrinsic regenerative capacity, which can be compromised by aging, disease, trauma, and iatrogenesis (e.g. tumor resection, pharmacological). At present, autografts and allografts are the principal biological treatments available to replace large bone segments, but both entail several limitations that reduce wider use and consistent success. The use of decellularized extracellular matrices (ECM), often derived from xenogeneic sources, has been shown to favorably influence the immune response to injury and promote site-appropriate tissue regeneration. Decellularized bone ECM (dbECM), utilized in several forms - whole organ, particles, hydrogels - has shown promise in both in vitro and in vivo animal studies to promote osteogenic differentiation of stem/progenitor cells and enhance bone regeneration. However, dbECM has yet to be investigated in clinical studies, which are needed to determine the relative efficacy of this emerging biomaterial as compared with established treatments. This mini-review highlights the recent exploration of dbECM as a biomaterial for skeletal tissue engineering and considers modifications on its future use to more consistently promote bone regeneration.

Role of NGF-TrkA signaling in calcification of articular chondrocytes.

Nerve growth factor (NGF) is a key regulator of chronic osteoarthritic pain, but the exact targets of NGF action on human articular cartilage is unknown. This study aimed to test the hypothesis that the NGF-tropomyosin receptor kinase A (TrkA) (high-affinity NGF receptor) pathway plays a role in the calcification process of human articular chondrocytes (hACs). A 14-aa small peptide of NGF (Nsp) previously shown to activate NGF signaling in rat PC12 cells was used as an NGF signaling agonist, and recombinant NGF and the pan-Trk inhibitor GNF-5837 were employed as signaling modulating agents. The functional consequences of NGF-TrkA signaling were examined in human healthy articular chondrocytes maintained under conditions supportive of osteogenesis in vitro. The NGF-mimetic bioactivity of Nsp was first confirmed on the basis of maintenance of neurite outgrowth in PC12 cells. Primary human chondrocytes responded to Nsp in vitro. Perturbation of NGF signaling with NGF, Nsp, and GNF-5837 resulted in a strong induction of chondrocyte calcification, and gene expression data suggested that the Indian Hedgehog-parathyroid hormone-related protein signaling axis was involved. These findings suggest functional involvement of NGF signaling in calcification of hACs and the importance of NGF signaling in articular cartilage homeostasis.-Jiang, Y., Tuan, R. S. Role of NGF-TrkA signaling in calcification of articular chondrocytes.

Lizard tail skeletal regeneration combines aspects of fracture healing and blastema-based regeneration.

Lizards are amniotes with the remarkable ability to regenerate amputated tails. The early regenerated lizard tail forms a blastema, and the regenerated skeleton consists of a cartilage tube (CT) surrounding the regenerated spinal cord. The proximal, but not distal, CT undergoes hypertrophy and ossifies. We hypothesized that differences in cell sources and signaling account for divergent cartilage development between proximal and distal CT regions. Exogenous spinal cord implants induced ectopic CT formation in lizard (Anolis carolinensis) blastemas. Regenerated spinal cords expressed Shh, and cyclopamine inhibited CT induction. Blastemas containing vertebrae with intact spinal cords formed CTs with proximal hypertrophic regions and distal non-hypertrophic regions, whereas removal of spinal cords resulted in formation of proximal CT areas only. In fate-mapping studies, FITC-labeled vertebra periosteal cells were detected in proximal, but not distal, CT areas. Conversely, FITC-labeled blastema cells were restricted to distal CT regions. Proximal cartilage formation was inhibited by removal of periosteum and could be recapitulated in vitro by periosteal cells treated with Ihh and BMP-2. These findings suggest that proximal CTs are directly derived from vertebra periosteal cells in response to BMP and Ihh signaling, whereas distal CTs form from blastema cells in response to Shh signals from regenerated spinal cords.

3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon-derived stem cells through a PI3K/AKT signaling pathway.

The tendon is a mechanosensitive tissue, but little is known about how mechanical stimulation selectively signals tenogenic differentiation and neo-tendon formation. In this study, we compared the impact of uniaxial and biaxial mechanical loading on tendon-derived stem cells (TDSCs). Our data show that there are variations in cell signaling and cell differentiation of mouse TDSCs in response to uniaxial and biaxial loading in monolayer culture. Whereas uniaxial loading induced TDSCs toward tenogenic and osteogenic differentiation, biaxial loading induced osteogenic, adipogenic, and chondrogenic differentiation of TDSCs. Furthermore, by applying uniaxial loading on 3-dimensional (3D) TDSC constructs, tenogenic-specific differentiation and neo-tendon formation were observed, results that were replicated in human TDSCs. We also showed that uniaxial loading induced PKB (AKT) phosphorylation (pAKT), whereas biaxial loading induced pERK. Most importantly, we found that inhibition of the PI3K/AKT signaling pathway could attenuate tenogenic differentiation and tendon formation in 3D TDSC constructs subjected to uniaxial loading. Taken together, our study highlights the importance of appropriate mechanobiological stimulation in 3D cell niches on tendon-like tissue formation and demonstrates that uniaxial mechanical loading plays an essential role in tenogenic differentiation and tendon formation by activating the PI3K/AKT signaling pathway.-Wang, T., Thien, C., Wang, C., Ni, M., Gao, J., Wang, A., Jiang, Q., Tuan, R. S., Zheng, Q., Zheng, M. H. 3D uniaxial mechanical stimulation induces tenogenic differentiation of tendon-derived stem cells through a PI3K/AKT signaling pathway.

The effect of adipose-derived stem cells on enthesis healing after repair of acute and chronic massive rotator cuff tears in rats.

BACKGROUND: Chronic massive rotator cuff tears heal poorly and often retear. This study investigated the effect of adipose-derived stem cells (ADSCs) and transforming growth factor-ß3 (TGF-ß3) delivered in 1 of 2 hydrogels (fibrin or gelatin methacrylate [GelMA]) on enthesis healing after repair of acute or chronic massive rotator cuff tears in rats. METHODS: Adult male Lewis rats underwent bilateral transection of the supraspinatus and infraspinatus tendons with intramuscular injection of botulinum toxin A (n = 48 rats). After 8 weeks, animals received 1 of 8 interventions (n = 12 shoulders/group): (1) no repair, (2) repair only, or repair augmented with (3) fibrin, (4) GelMA, (5) fibrin + ADSCs, (6) GelMA + ADSCs, (7) fibrin + ADSCs + TGF-ß3, or (8) GelMA + ADSCs + TGF-ß3. An equal number of animals underwent acute tendon transection and immediate application of 1 of 8 interventions. Enthesis healing was evaluated 4 weeks after the repair by microcomputed tomography, histology, and mechanical testing. RESULTS: Increased bone loss and reduced structural properties were seen in chronic compared with acute tears. Bone mineral density of the proximal humerus was higher in repairs of chronic tears augmented with fibrin + ADSCs and GelMA + ADSCs than in unrepaired chronic tears. Similar improvement was not seen in acute tears. No intervention enhanced histologic appearance or structural properties in acute or chronic tears. CONCLUSIONS: Surgical repair augmented with ADSCs may provide more benefit in chronic tears compared with acute tears, although there was no added benefit to supplementing ADSCs with TGF-ß3.

A Cellularized Biphasic Implant Based on a Bioactive Silk Fibroin Promotes Integration and Tissue Organization during Osteochondral Defect Repair in a Porcine Model.

In cartilage tissue engineering, biphasic scaffolds (BSs) have been designed not only to influence the recapitulation of the osteochondral architecture but also to take advantage of the healing ability of bone, promoting the implant's integration with the surrounding tissue and then bone restoration and cartilage regeneration. This study reports the development and characterization of a BS based on the assembly of a cartilage phase constituted by fibroin biofunctionalyzed with a bovine cartilage matrix, cellularized with differentiated autologous pre-chondrocytes and well attached to a bone phase (decellularized bovine bone) to promote cartilage regeneration in a model of joint damage in pigs. BSs were assembled by fibroin crystallization with methanol, and the mechanical features and histological architectures were evaluated. The scaffolds were cellularized and matured for 12 days, then implanted into an osteochondral defect in a porcine model (n = 4). Three treatments were applied per knee: Group I, monophasic cellular scaffold (single chondral phase); group II (BS), cellularized only in the chondral phase; and in order to study the influence of the cellularization of the bone phase, Group III was cellularized in chondral phases and a bone phase, with autologous osteoblasts being included. After 8 weeks of surgery, the integration and regeneration tissues were analyzed via a histology and immunohistochemistry evaluation. The mechanical assessment showed that the acellular BSs reached a Young's modulus of 805.01 kPa, similar to native cartilage. In vitro biological studies revealed the chondroinductive ability of the BSs, evidenced by an increase in sulfated glycosaminoglycans and type II collagen, both secreted by the chondrocytes cultured on the scaffold during 28 days. No evidence of adverse or inflammatory reactions was observed in the in vivo trial; however, in Group I, the defects were not reconstructed. In Groups II and III, a good integration of the implant with the surrounding tissue was observed. Defects in group II were fulfilled via hyaline cartilage and normal bone. Group III defects showed fibrous repair tissue. In conclusion, our findings demonstrated the efficacy of a biphasic and bioactive scaffold based on silk fibroin and cellularized only in the chondral phase, which entwined chondroinductive features and a biomechanical capability with an appropriate integration with the surrounding tissue, representing a promising alternative for osteochondral tissue-engineering applications.

Lizard tail regeneration as an instructive model of enhanced healing capabilities in an adult amniote.

The ability to regenerate damaged or lost tissues has remained the lofty goal of regenerative medicine. Unfortunately, humans, like most mammals, suffer from very minimal natural regenerative capabilities. Certain non-mammalian animal species, however, are not so limited in their healing capabilities, and several have attracted the attention of researchers hoping to recreate enhanced healing responses in humans. This review focuses on one such animal group with remarkable regenerative abilities, the lizards. As the closest relatives of mammals that exhibit enhanced regenerative abilities as adults, lizards potentially represent the most relevant model for direct comparison and subsequent improvement of mammalian healing. Lizards are able to regenerate amputated tails and exhibit adaptations that both limit tissue damage in response to injury and initiate coordinated regenerative responses. This review summarizes the salient aspects of lizard tail regeneration as they relate to the overall regenerative process and also presents the relevant information pertaining to regrowth of specific tissues, including skeletal, muscular, nervous, and vascular tissues. The goal of this review is to introduce the topic of lizard tail regeneration to new audiences with the hope of expanding the knowledge base of this underutilized but potentially powerful model organism.

Rapidly dissociated autologous meniscus tissue enhances meniscus healing: An in vitro study.

PURPOSE: Treatment of meniscus tears is a persistent challenge in orthopedics. Although cell therapies have shown promise in promoting fibrocartilage formation in in vitro and preclinical studies, clinical application has been limited by the paucity of autologous tissue and the need for ex vivo cell expansion. Rapid dissociation of the free edges of the anterior and posterior meniscus with subsequent implantation in a meniscus lesion may overcome these limitations. The purpose of this study was to explore the effect of rapidly dissociated meniscus tissue in enhancing neotissue formation in a radial meniscus tear, as simulated in an in vitro explant model. MATERIALS AND METHODS: All experiments in this study, performed at minimum with biological triplicates, utilized meniscal tissues from hind limbs of young cows. The effect of varying collagenase concentration (0.1%, 0.2% and 0.5% w/v) and treatment duration (overnight and 30 minutes) on meniscus cell viability, organization of the extracellular matrix (ECM), and gene expression was assessed through a cell metabolism assay, microscopic examination, and quantitative real-time reverse transcription polymerase chain reaction analysis, respectively. Thereafter, an explant model of a radial meniscus tear was used to evaluate the effect of a fibrin gel seeded with one of the following: (1) fibrin alone, (2) isolated and passaged (P2) meniscus cells, (3) overnight digested tissue, and (4) rapidly dissociated tissue. The quality of in vitro healing was determined through histological analysis and derivation of an adhesion index. RESULTS: Rapid dissociation in 0.2% collagenase yielded cells with higher levels of metabolism than either 0.1% or 0.5% collagenase. When seeded in a three-dimensional fibrin hydrogel, both overnight digested and rapidly dissociated cells expressed greater levels of collagens type I and II than P2 meniscal cells at 1 week. At 4 and 8 weeks, collagen type II expression remained elevated only in the rapid dissociation group. Histological examination revealed enhanced healing in all cell-seeded treatment groups over cell-free fibrin controls at weeks 1, 4, and 8, but there were no significant differences across the treatment groups. CONCLUSIONS: Rapid dissociation of meniscus tissue may provide a single-step approach to augment regenerative healing of meniscus repairs.

From embryonic development to human diseases: The functional role of caveolae/caveolin.

Caveolae, an almost ubiquitous, structural component of the plasma membrane, play a critical role in many functions essential for proper cell function, including membrane trafficking, signal transduction, extracellular matrix remodeling, and tissue regeneration. Three main types of caveolin proteins have been identified from caveolae since the discovery of caveolin-1 in the early 1990s. All three (Cav-1, Cav-2, and Cav-3) play crucial roles in mammalian physiology, and can effect pathogenesis in a wide range of human diseases. While many biological activities of caveolins have been uncovered since its discovery, their role and regulation in embryonic develop remain largely poorly understood, although there is increasing evidence that caveolins may be linked to lung and brain birth defects. Further investigations are clearly needed to decipher how caveolae/caveolins mediate cellular functions and activities of normal embryogenesis and how their perturbations contribute to developmental disorders.

Prenatal exposure to environmental factors and congenital limb defects.

Limb congenital defects afflict approximately 0.6:1000 live births. In addition to genetic factors, prenatal exposure to drugs and environmental toxicants, represents a major contributing factor to limb defects. Examples of well-recognized limb teratogenic agents include thalidomide, warfarin, valproic acid, misoprostol, and phenytoin. While the mechanism by which these agents cause dymorphogenesis is increasingly clear, prediction of the limb teratogenicity of many thousands of as yet uncharacterized environmental factors (pollutants) remains inexact. This is limited by the insufficiencies of currently available models. Specifically, in vivo approaches using guideline animal models have inherently deficient predictive power due to genomic and anatomic differences that complicate mechanistic comparisons. On the other hand, in vitro two-dimensional (2D) cell cultures, while accessible for cellular and molecular experimentation, do not reflect the three-dimensional (3D) morphogenetic events in vivo nor systemic influences. More robust and accessible models based on human cells that accurately replicate specific processes of embryonic limb development are needed to enhance limb teratogenesis prediction and to permit mechanistic analysis of the adverse outcome pathways. Recent advances in elucidating mechanisms of normal development will aid in the development of process-specific 3D cell cultures within specialized bioreactors to support multicellular microtissues or organoid constructs that will lead to increased understanding of cell functions, cell-to-cell signaling, pathway networks, and mechanisms of toxicity. The promise is prompting researchers to look to such 3D microphysiological systems to help sort out complex and often subtle interactions relevant to developmental malformations that would not be evident by standard 2D cell culture testing. Birth Defects Research (Part C) 108:243-273, 2016. © 2016 Wiley Periodicals, Inc.