Fat-derived Stem Cells Research Articles

THE CHALLENGES OF STEM CELL THERAPY FOR THE TREATMENT OF TENDINOPATHY IN THE CLINIC: LESSONS FROM A LARGE ANIMAL MODEL

Stem cells represent an exciting biological therapy for the management of many musculoskeletal tissues that suffer degenerative disease and/or where the reparative process results in non-functional tissue (‘failed healing’). The original hypothesis was that implanted cells would differentiate into the target tissue cell type and synthesise new matrix. However, this has been little evidence that this happens in live animals compared to the laboratory, and more recent theories have focussed on the immunomodulatory effects via the release of paracrine factors that can still improve the outcome, especially since inflammation is now considered one of the central processes that drive poor tendon healing. Because of the initial ‘soft’ regulatory environment for the use of stem cells in domestic mammals, bone and fat-derived stem cells quickly established themselves as a useful treatment for naturally occurring musculoskeletal diseases in the horse more than 20 years ago (Smith, Korda et al. 2003). Since the tendinopathy in the horse has many similarities to human tendinopathy, we propose that the following challenges and, the lessons learnt, in this journey are highly relevant to the development of stem cells therapies for human tendinopathy:Source – while MSCs can be recovered from many tissues, the predominant sources for autologous MSCs have been bone and fat. Other sources, including blood, amnion, synovium, and dental pulp have also been commercialised for allogenic treatments.Preparation – ex vivo culture requires transport from a licensed laboratory while ‘minimally manipulated’ preparations can be prepared patient-side. Cells also need a vehicle for transport and implantation.Delivery – transport of cells from the laboratory to the clinic for autologous ex vivo culture techniques; implantation technique (usually by ultrasound-guided injection to minimise damage to the cells (or, more rarely, incorporated into a scaffold). They can also be delivered by regional perfusion via venous or arterial routes.Retention – relatively poor although small numbers of cells do survive for at least 5 months. Immediate loss to the lungs if the cells are administered via vascular routes. Synovially administered cells do not engraft into tendon.Adverse effects – very safe although needle tracts often visible (but do not seen to adversely affect the outcome). Allogenic cells require careful characterisation for MHC Class II antigens to avoid anaphylaxis or reduced efficacy.Appropriate injuries to treat – requires a contained lesion when administered via intra-lesional injection. Intrasynovial tendon lesions are more often associated with surface defects and are therefore less appropriate for treatment. Earlier treatment appears to be more effective than delayed, when implantation by injection is more challenging.Efficacy - beneficial effects shown at both tissue and whole animal (clinical outcome) level in naturally-occurring equine tendinopathy using bone marrow-derived autologous MSCs Recent (licenced) allogenic MSC treatment has shown equivalent efficacy while intra-synovial administration of MSCs is ineffective for open intra-synovial tendon lesions.Regulatory hurdles – these have been lighter for veterinary treatments which has facilitated their development. There has been greater regulation of commercial allogenic MSC preparations which have required EMA marketing authorisation.

STIMULATING FRACTURE HEALING USING AUTOLOGOUS STEM CELLS IN A PRE-CLINICAL SYSTEM

To test and evaluate the effectiveness of local injection of autologous fat-derived mesenchymal stem cells (MSCs) into fracture site to prevent non-union in a clinically relevant model.5 male Wistar rats underwent the same surgical procedure of inducing non-union. A mid-shaft tibial osteotomy was made with 1mm non-critical gap. Periosteum was stripped around the two fracture ends. Then, the fracture was fixed by ante-grade intramedullary nail. The non-critical gap was maintained by a spacer with minimal effect on the healing surface area. At the same surgical time, subcutaneous fat was collected from the ipsilateral inguinal region and stem cells were isolated and cultured in vitro. Within three weeks postoperatively, the number of expanded stem cells reached 5×106 and were injected into the fracture site. Healing was followed up for 8 weeks and the quality was measured by serial x-rays, microCT, mechanical testing and histologically. Quality of healing was compared with that of previously published allogenic, xenogeneic MSCs and Purified Buffered Saline (PBS) controls.All the five fractures united fully after 8 weeks. There was a progressive increase in the callus radiopacity during the eight-week duration, the average radiopacity in the autologous fat-MSC injected group was significantly higher than that of the allogeneic MSCs, xenogeneic MSCs and the control group, P < 0.0001 for treatment, time after injection, and treatment-time interaction (two-way repeated measure ANOVA). MicroCT, mechanical testing and histology confirmed radiological findings.The autologous fat-MSCs are effective in prevention of atrophic non-union by stimulation of the healing process leading to a solid union. The quality and speed of repair are higher than those of the other types of cell transplantation tested.

Exosomal miR-103a-3p from Crohn's Creeping Fat-Derived Adipose-Derived Stem Cells Contributes to Intestinal Fibrosis by Targeting TGFBR3 and Activating Fibroblasts.

Mesenteric adipose tissue hypertrophy is a hallmark of Crohn's disease [CD], and creeping fat [CF] is unique to CD. Adipose-derived stem cells [ASCs] from inflammatory tissue exhibited altered biological functions. The role of ASCs isolated from CF in intestinal fibrosis and the potential mechanism remain unclear. ASCs were isolated from CF [CF-ASCs] and disease-unaffected mesenteric adipose tissue [Ctrl-ASCs] of patients with CD. A series of in vitro and in vivo experiments were conducted to study the effects of exosomes from CF-ASCs [CF-Exos] on intestinal fibrosis and fibroblast activation. A micro-RNA microarray analysis was performed. Western blot, luciferase assay and immunofluorescence were performed to further detect the underlying mechanisms. The results indicated that CF-Exos promoted intestinal fibrosis by activating fibroblasts in a dose-dependent manner. They continuously promoted progression of intestinal fibrosis even after dextran sulphate sodium withdrawal. Further analysis showed that exosomal miR-103a-3p was enriched in CF-Exos and participated in exosome-mediated fibroblast activation. TGFBR3 was identified as a target gene of miR-103a-3p. Mechanistically, CF-ASCs released exosomal miR-103a-3p and promoted fibroblast activation by targeting TGFBR3 and promoting Smad2/3 phosphorylation. We also found that the expression of miR-103a-3p in diseased intestine was positively associated with the degree of CF and fibrosis score. Our findings show that exosomal miR-103a-3p from CF-ASCs promotes intestinal fibrosis by activating fibroblasts via TGFBR3 targeting, suggesting that CF-ASCs are potential therapeutic targets for intestinal fibrosis in CD.

Breast Fat Grafting: 10-Year Learned Clinical Experience

Our being strives for staying youthful and being beautiful. Beauty for woman encompasses the entire body from the face, down to the breasts, the torso, buttocks, and extremities. Breast enhancement using implants is consistently ranked as one of the most common aesthetic surgical procedure undertaken by woman. However, as alloplastic implants suffer numerous potential complications and revision surgery, some practitioners and patients seek alternative treatment. Autologous fat grafting has gained popularity due to its safety and scientific advances yielding increased adipocyte survival. The low complication rate and valuable cosmetic outcomes will continue to have patients request natural breast augmentation. The authors’ more than 12 years of clinical experience with autologous fat transfer to the breast is presented. Advances in fat processing, enrichment, and administration are highlighted. The addition of stem cells, stromal vascular fraction, and platelet-rich plasma (PRP) is elucidated during the clinical experience. Laboratory evaluation of fat survival comparing ultrasound- and suction-assisted fat harvesting is presented. A comparison of 2 distinct fat processing methods is done using both clinical evaluation by the patients and surgeon, and the use of diagnostic ultrasound to assess fat thickness before and after surgical placement. The filtration device (Puregraft, Solana Beach, California) was compared with the Lipokit or filtration, centrifugation device (Medi-Khan USA, Inc., Los Angeles, California). Breast fat grafting was used for sole breast augmentation, after silicone breast implant surgery, in conjunction with mammopexy surgery, and reconstruction after breast cancer surgery or explantation. The authors’ observation with breast fat grafting volumetric outcomes has seen an increase from about 30% to almost 80%. This increased volume is related to the placement of a purer fat graft with less wetting solution fluid with less blood and oil. Minimizing ultrasound energy during the harvesting, lower suction levels, short fat storage times of 1 to 2 hours prior to administration, purification of fat without washing away growth factors, and the enrichment of fat with fat-derived autologous stem cells and blood-derived PRP have been observed techniques to optimize fat survival. Breast fat grafting using much of the current knowledge of harvesting, processing, enrichment, and administration has yielded superior adipocyte survival. This increased survival has provided more consistent breast enhancement results.

Efficacy and Safety of Fat-Derived Autologous Expanded Mesenchymal Stem Cell Therapy for the Management of Musculoskeletal Conditions: A 5-Year Experience

This editorial addresses the efficacy of single or repeated fat-derived autologous expanded Mesenchymal Stem Cell (MSC) therapy for orthopaedic conditions, specifically degenerative and injury-related joint and soft tissue disorders.

Ophthalmic Complications and Managements of Sulfur Mustard Exposure: A Narrative Review.

Sulfur mustard (SM) is a lethal chemical agent that affects many organs, particularly the eyes, respiratory system and skin. Even asymptomatic patients with documented SM vapor exposure may develop organ disorder many years later. Patients with even minor signs in the acute stage may experience late complications that necessitate surgery. Early decontamination and conservative measures could help the patients and decrease the complications. Despite decades of research, there is still no effective treatment for either acute or long-term SM-induced ocular complications. Even after multiple medications and surgical procedures, the majority of patients continue to have symptoms. For dry eye, punctual occlusion, autologous eye drops, and aggressive lubrication are used; for persistent epithelial defects (PED), tarsorrhaphy, amniotic membrane transplant, and stem cell transplantation are used; for total limbal stem cell deficiency (LSCD), living-related conjunctivolimbal allograft and keratolimbal allograft are used; for corneal vascularization, steroids, non-steroidal anti-inflammatory drugs, and anti-vascular endothelial growth factor prescribed; and for corneal opacities, corneal transplantation is done. Platelet rich plasma and topical drops containing stem cell transplantation for LSCD, photodynamic therapy paired with subconjunctival or topical anti-vascular endothelial growth factors for corneal vascularization, topical curcumin and topical ciclosporin-A for dry eye, and orbital fat-derived stem cells for PED are all alternative treatments that can be suggested. Despite the experimental and clinical research on the complications of SM exposure over the past decades, there is still no effective treatment for eye complications. However, supportive medical and surgical management has been applied with relatively good outcome.

Remarkable pain control and functional improvement in clubfeet-related ankle osteoarthritis post autologous expanded mesenchymal stem cell therapy

Ankle joint osteoarthritis (OA) is not as common as knee and hip OA but it causes a significant socioeconomic burden in society. The causes of ankle OA could be primary or secondary to previous ankle trauma, inflammatory arthritis like rheumatoid arthritis, avascular necrosis, post-infectious, clubfoot, and other rare causes. Ankle OA is hard to treat conservatively. Limited options, including physiotherapy, supportive custom-made braces, weight reduction, and anti-inflammatory medicines. Standard surgical options are joint arthrodesis and ankle replacement. Arthrodesis can limit the movement of talar and subtalar joints, making it difficult to be accepted by athletes who require functional joint movement. Ankle replacement might last between five and ten years and is sometimes technically challenging. Herein we report a case of a seventy-one-year-old male property investor. His past medical history of bilateral congenital clubfeet with the left foot more affected and pointed backward required three surgeries at the age of two through five to align the foot in the correct direction, and his left ankle was fused. In his mid-forties, he had mild ankles discomfort which was managed by suitable orthotics, despite the ankle pathology he enjoys various recreational physical sports at a premium competitive level with determination. At the age of sixty-seven, his symptoms increased gradually. He was diagnosed with severe secondary severe subtalar and mild talar OA, confirmed by X-rays; by the age of seventy-one, he had significant pain and loss of ankle function to the extent he could hardly walk five steps or play any sport. He failed conservative management with various supportive orthotics and long-term anti-inflammatory medicines. He elected experimental autologous fat-derived expanded mesenchymal stem cells (MSCs) combined with platelet-rich plasma (PRP). After two doses nine months apart, he responded superbly to become pain-free. Three months after the second dose, he was able to walk normally, play sports, and intend to return to competitive table tennis, but we recommended continuing using supportive orthotics. We predict MSCs slow the progression of OA. We believe this is the future medicine to try efficiently and safely regardless of the number of booster doses given.

Induction of Corneal Epithelial Differentiation of Induced Pluripotent and Orbital Fat-Derived Stem Cells Seeded on Decellularized Human Corneas.

Up to 40% of donor corneas are deemed unsuitable for transplantation, aggravating the shortage of graft tissue. In most cases, the corneal extracellular matrix is intact. Therefore, their decellularization followed by repopulation with autologous cells may constitute an efficient alternative to reduce the amount of discarded tissue and the risk of immune rejection after transplantation. Although induced pluripotent (hiPSCs) and orbital fat-derived stem cells (OFSCs) hold great promise for corneal epithelial (CE) reconstruction, no study to date has evaluated the capacity of decellularized corneas (DCs) to support the attachment and differentiation of these cells into CE-like cells. Here, we recellularize DCs with hiPSCs and OFSCs and evaluate their differentiation potential into CE-like cells using animal serum-free culture conditions. Cell viability and adhesion on DCs were assessed by calcein-AM staining and scanning electron microscopy. Cell differentiation was evaluated by RT-qPCR and immunofluorescence analyses. DCs successfully supported the adhesion and survival of hiPSCs and OFSCs. The OFSCs cultured under differentiation conditions could not express the CE markers, TP63, KRT3, PAX6, and KRT12, while the hiPSCs gave rise to cells expressing high levels of these markers. RT-qPCR data suggested that the DCs provided an inductive environment for CE differentiation of hiPSCs, supporting the expression of PAX6 and KRT12 without the need for any soluble induction factors. Our results open the avenue for future studies regarding the in vivo effects of DCs as carriers for autologous cell transplantation for ocular surface reconstruction.

Isolation and Characterization of Extracellular Vesicle from Mesenchymal Stem Cells of the Epidural Fat of the Spine.

Study DesignAn experimental study with extracellular vesicles (EVs) from mesenchymal stem cell (MSC) of the epidural fat (EF) of the spine.PurposeThis study aims to isolate the exosomes from epidural fat-derived mesenchymal stem cells (EF-MSCs) and fully characterize the EF-MSC-EVs.Overview of LiteratureEF-MSCs were reported in 2019, and a few studies have shown the positive outcomes of using EF-MSCs to treat specific spine pathologies. However, MSCs have significant limitations for conducting basic studies or developing therapeutic agents. Although EVs are an emerging research topic, no studies have focused on EVs, especially exosomes, from EF and EF-MSCs.MethodsIn this study, we isolated the exosomes using the tangential flow filtration (TFF) system with exosome-depleted fetal bovine serum and performed the characterization tests via western blotting, reverse transcription–polymerase chain reaction, nanoparticle tracking analysis (NTA), and transmission electron microscopy.ResultsIn transmission electron microscopy, the exosome had a diameter of approximately 100–200 nm and had a spherical shape, whereas in the NTA, the exosome had an average diameter of 142.8 nm with a concentration of 1.27×1010 particles/mL. The flow cytometry analysis showed the expression of CD63 and CD81. The western blotting analysis showed the positive markers.ConclusionsThese findings showed that isolating the exosomes via TFF resulted in high-quality EF-MSC exosome yield. Further studies with exosomes from EF-MSC are needed to evaluate the function and role of the EF tissue.

Is Fat Tissue a Suitable Source of Stem Cells for Musculoskeletal Regenerative Medicine Therapies: Literature Review

This review found that adipose-derived stem cells are suitable for treating osteoarthritis. Besides joint replacement the current treatments do not reverse the underlining pathology, whereas this therapy replaces lost or damaged tissue and could represent a cure. Bone marrow is the traditional source of stem cells, however adipose tissue is more appropriate because of ease of harvesting, abundance, the concentration of stem cells and potential cosmetic benefits. Subcutaneous fat-derived stem cells are more effective for treating osteoarthritis when compared to visceral fat-derived. Human and animal models have shown its efficacy but with limitations. It has been less successful in older and/or patients with greater joint degeneration. Its cost-effectiveness when compared to current treatment has not been shown. A gold standard production and implantation method have yet to be developed, meaning there are no guidelines for its use. Current studies aim to address these issues, with implementation on the horizon.

Comparisons of Extracellular Vesicles from Human Epidural Fat-Derived Mesenchymal Stem Cells and Fibroblast Cells.

Extracellular vesicles (EVs) are generated and secreted by cells into the circulatory system. Stem cell-derived EVs have a therapeutic effect similar to that of stem cells and are considered an alternative method for cell therapy. Accordingly, research on the characteristics of EVs is emerging. EVs were isolated from human epidural fat-derived mesenchymal stem cells (MSCs) and human fibroblast culture media by ultracentrifugation. The characterization of EVs involved the typical evaluation of cluster of differentiation (CD antigens) marker expression by fluorescence-activated cell sorting, size analysis with dynamic laser scattering, and morphology analysis with transmission electron microscopy. Lastly, the secreted levels of cytokines and chemokines in EVs were determined by a cytokine assay. The isolated EVs had a typical size of approximately 30–200 nm, and the surface proteins CD9 and CD81 were expressed on human epidural fat MSCs and human fibroblast cells. The secreted levels of cytokines and chemokines were compared between human epidural fat MSC-derived EVs and human fibroblast-derived EVs. Human epidural fat MSC-derived EVs showed anti-inflammatory effects and promoted macrophage polarization. In this study, we demonstrated for the first time that human epidural fat MSC-derived EVs exhibit inflammatory suppressive potency relative to human fibroblast-derived EVs, which may be useful for the treatment of inflammation-related diseases.

Resolution of Patellofemoral Bone Marrow Edema and Subchondral Cysts on MRI Post a Single Autologous Fat-Derived Expanded Mesenchymal Stem Cell Therapy

Resolution of Patellofemoral Bone Marrow Edema and Subchondral Cysts on MRI Post a Single Autologous Fat-Derived Expanded Mesenchymal Stem Cell Therapy

Three-dimensional environment and vascularization induce osteogenic maturation of human adipose-derived stem cells comparable to that of bone-derived progenitors.

While human adipose‐derived stem cells (hADSCs) are known to possess osteogenic differentiation potential, the bone tissues formed are generally considered rudimentary and immature compared with those made by bone‐derived precursor cells such as human bone marrow‐derived mesenchymal stem cells (hBMSCs) and less commonly studied human calvarium osteoprogenitor cells (hOPs). Traditional differentiation protocols have tended to focus on osteoinduction of hADSCs through the addition of osteogenic differentiation media or use of stimulatory bioactive scaffolds which have not resulted in mature bone formation. Here, we tested the hypothesis that by reproducing the physical as well as biochemical bone microenvironment through the use of three‐dimensional (3D) culture and vascularization we could enhance osteogenic maturation in hADSCs. In addition to biomolecular characterization, we performed structural analysis through extracellular collagen alignment and mineral density in our bone tissue engineered samples to evaluate osteogenic maturation. We further compared bone formed by hADSCs, hBMSCs, and hOPs against mature human pediatric calvarial bone, yet not extensively investigated. Although bone generated by all three cell types was still less mature than native pediatric bone, a fibrin‐based 3D microenvironment together with vascularization boosted osteogenic maturation of hADSC making it similar to that of bone‐derived osteoprogenitors. This demonstrates the important role of vascularization and 3D culture in driving osteogenic maturation of cells easily available but constitutively less committed to this lineage and suggests a crucial avenue for recreating the bone microenvironment for tissue engineering of mature craniofacial bone tissues from pediatric hADSCs, as well as hBMSCs and hOPs.

Differential expression of microRNA and arachidonic acid metabolism in aspirin-treated human cardiac and peri-cardiac fat-derived mesenchymal stem cells

Aspirin is a widely used drug with anti-coagulating and anti-inflammatory effects on atherosclerotic vascular disease. The goal of this study was to investigate expression of microRNA (miR) in association with changes in arachidonic acid (AA) metabolism in cardiac and surrounding fat mesenchymal stem cells (MSCs) treated with or without aspirin. Aspirin-targeted endogenous lipid metabolites that impact specific miRNA expression were examined by mass spectrometry. The pattern of miR expression was characterized using a microarray of 1100 miRs. There were a dozen miRs expressed differentially in MSCs from human myocardium and peri-myocardial fat tissue at baseline, including hsa-miR-1307-3p, 765, 4739, 3613-3p, 4281, 6816-5p, 2861, and 146b-5p. After exposure to aspirin, cardiac MSCs expressed an array of of miRs (eg, hsa-miR-4734, 10a-5p, 4267, 3197, and 3182), while generation of their endogenous AA metabolites was depressed. However, in the peri-cardiac adipose tissue-derived MSCs, treatment with the same doses of aspirin caused mild changes in the miR expression levels. In conclusion, MSCs from human myocardium and peri-myocardial fat tissue respond differentially to aspirin treatment by alterations in miR expression and AA metabolism. The study further raises an intriguing issue as to whether the copious amounts of aspirin taken worldwide by patients with cardiovascular disease may have direct impacts on their heart repair processes by regulation of stromal cell miR expression and AA metabolism.

Human Fat-Derived Mesenchymal Stem Cells Xenogenically Implanted in a Rat Model Show Enhanced New Bone Formation in Maxillary Alveolar Tooth Defects.

Background Due to restorative concerns, bone regenerative therapies have garnered much attention in the field of human oral/maxillofacial surgery. Current treatments using autologous and allogenic bone grafts suffer from inherent challenges, hence the ideal bone replacement therapy is yet to be found. Establishing a model by which MSCs can be placed in a clinically acceptable bone defect to promote bone healing will prove valuable to oral/maxillofacial surgeons. Methods Human adipose tissue-derived MSCs were seeded onto Gelfoam® and their viability, proliferation, and osteogenic differentiation was evaluated in vitro. Subsequently, the construct was implanted in a rat maxillary alveolar bone defect to assess in vivo bone healing and regeneration. Results Human MSCs were adhered, proliferated, and uniformly distributed, and underwent osteogenic differentiation on Gelfoam®, comparable with the tissue culture surface. Data confirmed that Gelfoam® could be used as a scaffold for cell attachment and a delivery vehicle to implant MSCs in vivo. Histomorphometric analyses of bones harvested from rats treated with hMSCs showed statistically significant increase in collagen/early bone formation, with cells positive for osteogenic and angiogenic markers in the defect site. This pattern was visible as early as 4 weeks post treatment. Conclusions Xenogenically implanted human MSCs have the potential to heal an alveolar tooth defect in rats. Gelfoam®, a commonly used clinical biomaterial, can serve as a scaffold to deliver and maintain MSCs to the defect site. Translating this strategy to preclinical animal models provides hope for bone tissue engineering.

Chyle Fat-Derived Stem Cells Conditioned Medium Inhibits Hypertrophic Scar Fibroblast Activity.

Hypertrophic scars (HSs) generally form after injury to the deep layers of the dermis and are characterized by excessive collagen deposition. An increasing amount of evidence has determined that human adipose tissue-derived mesenchymal stem cells attenuate fibrosis in various conditions. We explored the effect and possible mechanism of chyle fat-derived stem cells (CFSCs) on HS formation. Hypertrophic scar-derived fibroblasts (HSFs) and CFSCs were isolated from individual patients. Third-passage CFSCs were isolated and cultured using a mechanical emulsification method, and their surface CD markers were analyzed by flow cytometry. The adipogenic and osteogenic differentiation capacity of the CFSCs was determined using oil red O staining and alizarin red S staining, respectively. Then, the effects of CFSCs on HSFs were assessed in vitro. Hypertrophic scar-derived fibroblasts were treated with starvation-induced conditioned medium from the CFSCs (CFSC-CM). The change in HSF cellular behaviors, such as cell proliferation, migration, and protein expression of scar-related molecules, was evaluated by cell counting assay, scratch wound assay, enzyme-linked immunosorbent assay, and western blotting. All data were analyzed using SPSS 17.0. The CFSCs expressed CD90, CD105, and CD73 but did not express CD34, CD45, or CD31. The CFSCs differentiated into adipocytes and osteoblasts under the appropriate induction conditions. Chyle fat-derived stem cells conditioned medium inhibited HSF proliferation and migration. The in vitro and ex vivo studies revealed that CFSC-CM decreased type I collagen, type III collagen, and α smooth muscle actin expression. Our results suggest that CFSCs are associated with the inhibition of fibrosis in HSFs by a paracrine effect. The use of CFSC-CM may be a novel therapeutic strategy for HSs.

108. Posterior bone graft options and success in single-level circumferential lumbar fusions

BACKGROUND CONTEXT Achieving spinal fusion requires bone graft and is most reliable using an anterior/posterior technique. Anterior fusion, due to the large bony surface area between the vertebral bodies, results in reliable fusion. Instrumentation which provides stability, also increases the fusion rate. The bone graft for the posterior fusion may come from a variety of sources. Historically, bone graft was obtained from the iliac portion of the patient's own pelvis, IBG, but this may result in additional postoperative pain. Alternatives are currently the most common form of bone graft, but there is very little knowledge as to the superiority of one alternative over another. PURPOSE To determine the ability to achieve a solid posterior spinal fusion using sources of bone graft derived from IBG, bone morphogenic protein (BMP), or from donor bone combined with stem cells derived from either autologous bone marrow aspirate (BMA) or donor bone, fat, or amniotic membrane/fluid. STUDY DESIGN/SETTING Prospective randomized single-blinded clinical study of 6 comparative groups with 27 patients in each group. PATIENT SAMPLE Adults with advanced degenerative disc disease (DDD) who underwent single-level lumbar spinal fusion. OUTCOME MEASURES Outcomes included self-assessment of back and leg pain, pain drawing, ODI, pain medication usage, and procedure success. High resolution CT scans at one year postoperative assessed fusion rates between groups. Costs of graft materials were compared. METHODS Outcomes were obtained prospectively. Patients were enrolled over a 7-year period and followed for 2-4 years. All single-level circumferential fusions used BMP anteriorly, instrumentation, and patients were randomized into 6 groups with a different type of bone-graft used posteriorly. RESULTS CT scans found solid interbody fusion in 98% of patients. However, solid posterior fusions varied between groups: 98% IBG, 94% BMP, 78% concentrated BMA, 70% bone allograft stem cells, and 64% each for amniotic tissue and fat derived stem cells. Clinical improvement was significant from preop to 2-4 year follow-up within all groups (p CONCLUSIONS One-level circumferential spinal fusion using IBG was the most reliable and cost-effective graft material. BMP was highly effective and the most expensive. Concentrated autologous BMA had moderate fusion success and cost. Allograft products with varying cellularity were marginally effective for their high cost. FDA DEVICE/DRUG STATUS Unavailable from authors at time of publication.

Optimizing 3D Co-culture Models to Enhance Synergy Between Adipose-Derived Stem Cells and Chondrocytes for Cartilage Tissue Regeneration

Donor cell scarcity is a key barrier for clinical translation of chondrocytes for regenerating cartilage. To overcome this challenge, recent studies have utilized mixed populations of chondrocytes and adult stem cells to show synergistic interactions during 3D co-culture using hydrogels or pellets. Yet, how different co-culture models impact the synergy and resulting cartilage formation remains unknown. To determine the optimal delivery method of a mixed population of stem cells and chondrocytes for cartilage regeneration, here we compared hydrogel and pellet co-culture on the interactions between adipose-derived stem cells (ADSCs) and neonatal chondrocytes (NChons). While both co-culture models supported synergy, hydrogel co-culture led to over a 5-fold increase in synergistic index in cell proliferation, and over 11 and 15-fold increases in synergistic indices for production of cartilage matrix (collagen and sGAG respectively). Interaction index analyses of gene expressions showed hydrogel co-culture significantly reduced hypertrophic phenotype of both ADSCs and NChons. In hydrogel co-culture, NChons contributed to the neocartilage deposition while ADSCs resided outside the newly formed cartilage nodule, serving as supporting cells through paracrine signaling. In contrast, during pellet co-culture, both NChons and ADSCs contributed to the newly formed cartilage. Using the same number of cells, hydrogel co-culture supported higher synergy and produced neocartilage tissues approximately 5 times the size of pellet co-culture. In contrast, pellet co-culture resulted in denser cartilage matrix suitable only for small defects. The outcomes of this study suggest hydrogel delivery as a more advantageous co-culture method for regenerating cartilage using mixed cell populations. Donor cell scarcity is a key barrier for clinical translation of cell-based therapies for cartilage regeneration. To overcome this challenge, here we harnessed abundantly available fat-derived stem cells and mixed them with juvenile chondrocytes, a cell type from native cartilage that can produce robust cartilage. We compared two different co-culture methods, hydrogel versus cell pellet, to grow the mixed cell populations for cartilage regeneration. We demonstrate that 3D hydrogels induced higher synergy using mixed cell populations, allowing cartilage repair with a reduced number of chondrocytes and defect filling of larger volumes than pellets. Future work will validate the potential of using hydrogels to deliver mixed population of fat-derived stem cells and juvenile chondrocytes for cartilage regeneration in vivo using relevant animal models.

2039-P: Age-Related Dysfunction of Brown Adipose Progenitor Cells

Aging is a primary risk factor for the development of obesity and diabetes mellitus and is accompanied by impaired brown adipocyte formation. Deprivation of thermogenic capacity may contribute to an obesity-prone phenotype of aged individuals. To determine whether the age-related decline of brown adipogenesis is due to a cell-autonomous dysfunction of brown adipogenic cells, we isolated stem cells from brown adipose tissue of mice aged 2.5 or 25 months for characterization. While lipid accumulation capacity was unchanged, expression of brown adipocyte markers, such as Uncoupling protein-1 (Ucp1), were significantly reduced in aged cells after differentiation, suggesting that a cell-intrinsic defect is responsible for impaired brown adipogenesis during aging. We subsequently generated immortalized single stem cell clones from both age groups, which displayed a similar age-related defect. Transcriptomes generated from a total of 126 clones show that brown fat-derived stem cells from old mice more closely resemble stem cells isolated from white adipose tissue. Among the genetic pathways significantly altered in aged stem cells are changes to extracellular matrix function as well as cell-intrinsic pathways. Importantly, aged stem cells are characterized by an age-dependent induction of cellular senescence with increased expression of cell cycle inhibitors p16, p21 and p53. To further test the link between senescence and reduced brown adipogenic potential, we isolated brown adipogenic stem cells from a mouse model with conditional inactivation of p53. Confirming our hypothesis, p53-deficient stem cells displayed a marked increase in Ucp1-expression upon differentiation when compared to wild type stem cells. These data taken together suggest that aging of brown adipogenic stem cells may contribute to loss of brown adipocytes and that inhibition of cell cycle blockage may restore their ability to undergo full brown adipogenic differentiation. Disclosure A. Graja: None. S. Gohlke: None. L. Saraiva: None. T.J. Schulz: None. Funding German Center for Diabetes Research; German Research Foundation

Temporomandibular Disorders Clinical and Anatomical Outcomes After Fat-Derived Stem Cells Injection.

Temporomandibular disorders (TMD) are common disorders that usually involve temporomandibular joint (TMJ), masticatory muscles, and other relevant structures. The symptoms may vary limiting the patients' quality of life. Many treatment options were proposed during the last years with the aim to treat the pathology. In this article, we analyze the effect of the injection of the fat-derived stem cell in the joint as a new treatment option.