Isolation of the Stromal Vascular Fraction Using a New Protocol with All Clinical-Grade Drugs: From Basic Study to Clinical Application.

OBJECTIVE: The purpose of this study was to evaluate the yield, viability, clinical safety, and efficacy of the stromal vascular fraction (SVF) separated with a new protocol with all clinical-grade drugs. MATERIALS AND METHODS: SVF cells were isolated from lipoaspirate obtained from 13 participants aged from 30 to 56 years by using a new clinical protocol and the laboratory protocol. The cell yield, viability, morphology, mesenchymal stem cell (MSC) surface marker expression, and differentiation abilities of the SVF cells harvested from the two protocols were compared. Furthermore, three related clinical trials were conducted to verify the safety and efficiency of SVF cells isolated by the new clinical protocol. RESULTS: There were no significant differences in the yield, viability, morphology, and differentiation potential of the SVFs isolated with the clinical protocol and laboratory protocol. Adipose-derived mesenchymal stem cell (ASC) surface marker expression, including that of CD14, CD31, CD44, CD90, CD105, and CD133, was consistent between the two protocols. Clinical trials have demonstrated the effectiveness of the SVF isolated with the new clinical protocol in improving skin grafting, promoting mechanical stretch-induced skin regeneration and improving facial skin texture. No complications occurred. CONCLUSION: SVF isolated by the new clinical protocol had a noninferior yield and viability to that of the SVF separated by the laboratory protocol. SVFs obtained by the new protocol can be safely and effectively applied to improve skin grafting, promote mechanical stretch-induced skin regeneration, and improve facial skin texture. TRIAL REGISTRATION: The trials were registered with the ClinicalTrials.gov (NCT03189628), the Chinese Clinical Trial Registry (ChiCTR2000039317), and the ClinicalTrials.gov (NCT02546882). All the three trials were not patient-funded trials. NO LEVEL ASSIGNED: This journal requires that authors assign a level of evidence to each submission to which Evidence-Based Medicine rankings are applicable. This excludes Review Articles, Book Reviews, and manuscripts that concern Basic Science, Animal Studies, Cadaver Studies, and Experimental Studies. For a full description of these Evidence-Based Medicine ratings, please refer to the Table of Contents or the online Instructions to Authors www.springer.com/00266 .

Exploration of treatment in childhood Langerhans cell histiocytosis based on inflammatory and malignant symptoms: a pilot study.

BACKGROUND: Multisystem childhood Langerhans cell histiocytosis (LCH) patients, especially those with risk organ (RO) involved, had not been satisfactorily treated under the international traditional schemes as high incidences of reactivation with late sequelae were largely reported. Over years, we have observed that LCH patients with varied clinical symptoms responded differently to different drugs, suggesting the current grouping strategies based only on the number of organs involved might be inadequate. LCH has been defined as an inflammatory myeloid tumor, thus this study has innovatively divided LCH pediatric patients into inflammatory or malignant symptoms group, and given different intensity treatment regimens to different groups. AIM: This clinical study aimed to explore a more appropriate patient grouping system according to the LCH symptom presentations and examine the clinical outcomes of treatment strategies in different groups. METHODS: According to the clinical manifestations, 37 cases of children were divided into Group A (only inflammatory symptoms) and Group B (malignant symptoms with or without inflammatory symptoms). Patients in Group A and B were initially treated with vindesine (VDS) and methylprednisolone (PSL), and VDS, PSL, pirarubicin (THP) and cyclophosphamide (CTX), respectively. Treatment responses were evaluated six weeks after the induction therapy in all patients, and the criteria were disease status and clinical scores of symptoms. RESULTS: Pre- and post-treatment scores were 1.22 ± 0.547 and 0.00 ± 0.00 in Group A, and 14.79 ± 1.686 and 1.00 ± 1.563 in Group B, respectively. All patients had subsequentlly received maintenance therapy without progressive disease. The 4-year overall survival (OS) rate was 100% in both groups and the 4-year event-free survival (EFS) was 94.4% in Group A and 89.5% in Group B, respectively. There were no obvious adverse events (AE) in Group A, whereas the main AE in Group B were alopecia and non-lethal hematological toxicity. CONCLUSION: Stratification according to patients' clinical symptoms, with low-intensity treatment for inflammatory symptoms (mild manifestations) and intensive treatment with multiple drugs for malignant symptoms (severe manifestations), is a positive exploration that simplifies stratification method, achieves good long-term remission of the disease, and obtains a higher survival rate and quality of life, which seemed to be more appropriate for LCH patients.

Bioengineering autologous cartilage grafts for functional posterior lamellar eyelid reconstruction: A preliminary study in rabbits.

The reconstruction of posterior lamellar eyelid defects remains a significant challenge in clinical practice due to anatomical complexity, specialized function, and aesthetic concerns. The ideal substitute for the posterior lamellar should replicate the native tarsoconjunctival tissue, providing both mechanical support for the eyelids and a smooth surface for the globe after implantation. In this study, we present an innovative approach utilizing tissue-engineered cartilage (TEC) grafts generated from rabbit auricular chondrocytes and a commercialized type I collagen sponge to reconstruct critical-sized posterior lamellar defects in rabbits. The TEC grafts demonstrated remarkable mechanical strength and maintained a stable cartilaginous phenotype both in vitro and at 6 months post-implantation in immunodeficient mice. When employed as autografts to reconstruct tarsal plate defects in rabbits' upper eyelids, these TEC grafts successfully restored normal eyelid morphology, facilitated smooth eyelid movement, and preserved the histological structure of the conjunctival epithelium. When applied in bilayered tarsoconjunctival defect reconstruction, these TEC grafts not only maintained the normal contour of the upper eyelid but also supported conjunctival epithelial cell migration and growth from the defect margin towards the centre. These findings highlight that auricular chondrocyte-based TEC grafts hold great promise as potential candidates for clinical posterior lamellar reconstruction. STATEMENT OF SIGNIFICANCE: The complex structure and function of the posterior lamellar eyelid continue to be significant challenges for clinical reconstructive surgeries. In this study, we utilized autologous auricular chondrocyte-based TEC grafts for posterior lamellar eyelid reconstruction in a preclinical rabbit model. The TEC grafts exhibited native cartilaginous histomorphology and comparable mechanical strength to those of the native human tarsal plate. In rabbit models with either tarsal plate defects alone or bilayered tarsoconjunctival defects, TEC grafts successfully restored the normal eyelid contour and movement, as well as supported preservation and growth of conjunctival epithelium. This is the first study to demonstrate autologous TEC grafts can be employed for repairing tarsal plate defects, thereby offering an alternative therapeutic approach for treating posterior lamellar defects in clinic settings.

Unleashing the potential of adipose organoids: A revolutionary approach to combat obesity-related metabolic diseases.

Obesity-related metabolic diseases, including obesity, diabetes, hyperlipidemia, and non-alcoholic fatty liver diseases pose a significant threat to health. However, comprehensive pathogenesis exploration and effective therapy development are impeded by the limited availability of human models. Notably, advances in organoid technology enable the generation of adipose organoids that recapitulate structures and functions of native human adipose tissues to investigate mechanisms and develop corresponding treatments for obesity-related metabolic diseases. Here, we review the general principles, sources, and three-dimensional techniques for engineering adipose organoids, along with strategies to promote maturation. We also outline the application of white adipose organoids, primarily for disease modeling and drug screening, and highlight the therapeutic potential of thermogenic beige and brown adipose organoids in promoting weight loss and glucose and lipid metabolic homeostasis. We also discuss the challenges and prospects in the establishment and bench-to-bedside of adipose organoids, as well as their potential applications.

Bioengineering and vascularization strategies for islet organoids: advancing toward diabetes therapy.

Diabetes presents a pressing healthcare crisis, necessitating innovative solutions. Organoid technologies have rapidly advanced, leading to the emergence of bioengineering islet organoids as an unlimited source of insulin-producing cells for treating insulin-dependent diabetes. This advancement surpasses the need for cadaveric islet transplantation. However, clinical translation of this approach faces two major limitations: immature endocrine function and the absence of a perfusable vasculature compared to primary human islets. In this review, we summarize the latest developments in bioengineering functional islet organoids in vitro and promoting vascularization of organoid grafts before and after transplantation. We highlight the crucial roles of the vasculature in ensuring long-term survival, maturation, and functionality of islet organoids. Additionally, we discuss key considerations that must be addressed before clinical translation of islet organoid-based therapy, including functional immaturity, undesired heterogeneity, and potential tumorigenic risks.

Bioengineered human tissue regeneration and repair using endogenous stem cells.

We describe a general approach to produce bone and cartilaginous structures utilizing the self-regenerative capacity of the intercostal rib space to treat a deformed metacarpophalangeal joint and microtia. Anatomically precise 3D molds were positioned on the perichondro-periosteal or perichondral flap of the intercostal rib without any other exogenous elements. We find anatomically precise metacarpal head and auricle constructs within the implanted molds after 6 months. The regenerated metacarpal head was used successfully to surgically repair the deformed metacarpophalangeal joint. Auricle reconstructive surgery in five unilateral microtia patients yielded good aesthetic and functional results. Long-term follow-up revealed the auricle constructs were safe and stable. Single-cell RNA sequencing analysis reveal early infiltration of a cell population consistent with mesenchymal stem cells, followed by IL-8-stimulated differentiation into chondrocytes. Our results demonstrate the repair and regeneration of tissues using only endogenous factors and a viable treatment strategy for bone and tissue structural defects.

Biomaterials and tissue engineering strategies for posterior lamellar eyelid reconstruction: Replacement or regeneration?

Reconstruction of posterior lamellar eyelids remains challenging due to their delicate structure, highly specialized function, and cosmetic concerns. Current clinically available techniques for posterior lamellar reconstruction mainly focus on reconstructing the contour of the eyelids. However, the posterior lamella not only provides structural support for the eyelid but also offers a smooth mucosal surface to facilitate globe movement and secrete lipids to maintain ocular surface homeostasis. Bioengineered posterior lamellar substitutes developed via acellular or cellular approaches have shown promise as alternatives to current therapies and encouraging outcomes in animal studies and clinical conditions. Here, we provide a brief reference on the current application of autografts, biomaterials, and tissue-engineered substitutes for posterior lamellar eyelid reconstruction. We also shed light on future challenges and directions for eyelid regeneration strategies and offer perspectives on transitioning replacement strategies to regeneration strategies for eyelid reconstruction in the future.

Body fluid-derived stem cells - an untapped stem cell source in genitourinary regeneration.

Somatic stem cells have been obtained from solid organs and tissues, including the bone marrow, placenta, corneal stroma, periosteum, adipose tissue, dental pulp and skeletal muscle. These solid tissue-derived stem cells are often used for tissue repair, disease modelling and new drug development. In the past two decades, stem cells have also been identified in various body fluids, including urine, peripheral blood, umbilical cord blood, amniotic fluid, synovial fluid, breastmilk and menstrual blood. These body fluid-derived stem cells (BFSCs) have stemness properties comparable to those of other adult stem cells and, similarly to tissue-derived stem cells, show cell surface markers, multi-differentiation potential and immunomodulatory effects. However, BFSCs are more easily accessible through non-invasive or minimally invasive approaches than solid tissue-derived stem cells and can be isolated without enzymatic tissue digestion. Additionally, BFSCs have shown good versatility in repairing genitourinary abnormalities in preclinical models through direct differentiation or paracrine mechanisms such as pro-angiogenic, anti-apoptotic, antifibrotic, anti-oxidant and anti-inflammatory effects. However, optimization of protocols is needed to improve the efficacy and safety of BFSC therapy before therapeutic translation.

Botulinum Toxin Type A Injection for Correction of Upper Eyelid Retraction in Thyroid-Associated Ophthalmopathy.

Upper lid retraction (ULR) is the most common and earliest symptom in thyroid-associated ophthalmopathy (TAO) patients. Surgical correction is effective for ULR in stable diseases. However, non-invasive treatment is also required for the TAO patient in active phase. Here, we reported a complex case with TAO and unilateral ULR simultaneously. The patient had a history of progressive ptosis in the left eyelid and underwent anterior levator aponeurotic-Muller muscle resection to correct the ptosis. However, the patient gradually developed bilateral proptosis and ULR, mainly in the left eyelid. The patient was finally diagnosed with TAO with left ULR. Then, the patient was treated with botulinum toxin type A (BTX-A) injection in the left eyelid. The effect of BTX-A treatment started 7 days after injection, peaked at one month, and lasted for approximately 3 months. This study highlighted the therapeutic effect of BTX-A injection for the treatment of ULR-related TAO.

Engineered M13 phage as a novel therapeutic bionanomaterial for clinical applications: From tissue regeneration to cancer therapy.

Bacteriophages (phages) are nanostructured viruses with highly selective antibacterial properties that have gained attention beyond eliminating bacteria. Specifically, M13 phages are filamentous phages that have recently been studied in various aspects of nanomedicine due to their biological advantages and more compliant engineering capabilities over other phages. Having nanofiber-like morphology, M13 phages can reach varied target sites and self-assemble into multidimensional scaffolds in a relatively safe and stable way. In addition, genetic modification of the coat proteins enables specific display of peptides and antibodies on the phages, allowing for precise and individualized medicine. M13 phages have also been subjected to novel engineering approaches, including phage-based bionanomaterial engineering and phage-directed nanomaterial combinations that enhance the bionanomaterial properties of M13 phages. In view of these features, researchers have been able to utilize M13 phages for therapeutic applications such as drug delivery, biodetection, tissue regeneration, and targeted cancer therapy. In particular, M13 phages have been utilized as a novel bionanomaterial for precisely mimicking natural tissue environment in order to overcome the shortage in tissue and organ donors. Hence, in this review, we address the recent studies and advances of using M13 phages in the field of nanomedicine as therapeutic agents based upon their characteristics as novel bionanomaterial with biomolecules displayed. This paper also emphasizes the novel engineering approach that enhances M13 phage's bionanomaterial capabilities. Current limitations and future approaches are also discussed to provide insight in further progress for M13 phage-based clinical applications.

Autologous Concentrated Growth Factor Increases Skin Thickness and Area during Tissue Expansion: A Randomized Clinical Trial.

BACKGROUND: Mechanical stretching of the skin (ie, tissue expansion) could generate additional skin, but it is limited by the intrinsic growth capacity. The authors conducted a study of autologous concentrated growth factor (CGF) to promote skin regeneration by increasing skin thickness and area during tissue expansion. METHODS: A single-center randomized controlled trial was conducted from 2016 to 2019. Participants undergoing skin expansion received either CGF or saline by means of intradermal injection on the expanded skin (0.02 mL/cm 2 ), for a total of three treatments at 4-week intervals. The primary endpoint was the expanded skin thickness at 12 weeks, which was measured by ultrasound. The secondary endpoints included skin thickness at 4 and 8 weeks and surface area, expansion index, and skin texture score of the expanded skin at 12 weeks. Safety assessments, for infection symptoms and nodule formation, were assessed at 24 weeks. RESULTS: In total, 26 patients were enrolled and assigned to the CGF or control group. Compared with the control group, the CGF group had significantly increased skin thickness at 8 (control, 1.1 ± 0.1 mm; CGF, 1.4 ± 0.1 mm; -0.6 to 0.0 mm; P = 0.047) and 12 weeks (control, 1.0 ± 0.1 mm; CGF, 1.3 ± 0.1 mm; -0.6 to 0.0 mm; P = 0.047). Compared with the baseline thickness (control, 1.6 ± 0.1 mm; CGF, 1.5 ± 0.1 mm; -0.3 to 0.5 mm; P = 0.987), skin thickness was sustained in the CGF group at 8 weeks after treatment (-0.1 to 0.3 mm; P = 0.711) but decreased in the control group (0.3 to 0.7 mm; P < 0.001). At 12 weeks, the CGF group showed greater increases in surface area (control, 77.7 ± 18.5 cm 2 ; CGF, 135.0 ± 15.7 cm 2 ; 7.2 cm 2 to 107.4 cm 2 ; P = 0.027) and expansion index (control, 0.9 ± 0.1; CGF, 1.4 ± 0.2; 0.0 to 0.8; P = 0.030) than the control group. In addition, CGF-treated skin showed an improvement in texture [CGF: grade 3, n = 2 (15.8%), grade 2, n = 4 (30.7%); control: grade 3, n = 0 (0.0%), grade 2, n = 3 (23.0%)]. No severe adverse events occurred. CONCLUSION: CGF treatment increases skin thickness and area during tissue expansion, and represents a safe and effective strategy for managing skin expansion. CLINICAL RELEVANCE STATEMENT: The findings of this study indicate that it is practically feasible to improve skin regeneration by applying autologous platelet concentrate therapy for skin expansion management. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

Engineering hypertrophic cartilage grafts from lipoaspirate for critical-sized calvarial bone defect reconstruction: An adipose tissue-based developmental engineering approach.

Developmental engineering of living implants from different cell sources capable of stimulating bone regeneration by recapitulating endochondral ossification (ECO) is a promising strategy for large bone defect reconstruction. However, the clinical translation of these cell-based approaches is hampered by complex manufacturing procedures, poor cell differentiation potential, and limited predictive in vivo performance. We developed an adipose tissue-based developmental engineering approach to overcome these hurdles using hypertrophic cartilaginous (HyC) constructs engineered from lipoaspirate to repair large bone defects. The engineered HyC constructs were implanted into 4-mm calvarial defects in nude rats and compared with decellularized bone matrix (DBM) grafts. The DBM grafts induced neo-bone formation via the recruitment of host cells, while the HyC pellets supported bone regeneration via ECO, as evidenced by the presence of remaining cartilage analog and human NuMA-positive cells within the newly formed bone. However, the HyC pellets clearly showed superior regenerative capacity compared with that of the DBM grafts, yielding more new bone formation, higher blood vessel density, and better integration with adjacent native bone. We speculate that this effect arises from vascular endothelial growth factor and bone morphogenetic protein-2 secretion and mineral deposition in the HyC pellets before implantation, promoting increased vascularization and bone formation upon implantation. The results of this study demonstrate that adipose-derived HyC constructs can effectively heal large bone defects and present a translatable therapeutic option for bone defect repair.

Surgical Strategies for Eyelid Defect Reconstruction: A Review on Principles and Techniques.

Reconstruction of eyelid defects, especially the posterior lamella, remains challenging because of its anatomical complexity, functional considerations, and aesthetic concerns. The goals of eyelid reconstruction include restoring eyelid structure and function and achieving an aesthetically acceptable appearance. An in-depth understanding of the complex eyelid anatomy and several reconstructive principles are mandatory to achieve these goals. Currently, there are multiple surgical treatment options for eyelid reconstruction, including different flaps, grafts, and combinations of them. This comprehensive review outlines the principles of reconstruction and discusses the indications, advantages, and disadvantages of currently available surgical techniques. We also propose our clinical thinking for solving specific clinical questions in eyelid reconstruction and offer perspectives on new potential methodologies in the future.

Downregulation of PLK4 expression induces apoptosis and G0/G1-phase cell cycle arrest in keloid fibroblasts.

OBJECTIVES: Keloids are benign fibroproliferative tumors that display many cancer-like characteristics, such as progressive uncontrolled growth, lack of spontaneous regression, and extremely high rates of recurrence. Polo-like kinase 4 (PLK4) was recently identified as a master regulator of centriole replication, and its aberrant expression is closely associated with tumorigenesis. This study aimed to investigate the expression and biological role of PLK4 in the pathogenesis of keloids. MATERIALS AND METHODS: We evaluated the expression of PLK4 in keloids and adjacent normal skin tissue samples. Then, we established PLK4 knockdown and overexpression cell lines in keloid fibroblasts (KFs) and normal skin fibroblasts (NFs), respectively, to investigate the roles of PLK4 in the regulation of proliferation, migration, invasion, apoptosis, and cell cycle in KFs. Centrinone B (Cen-B), a highly selective PLK4 inhibitor, was used to inhibit PLK4 activity in KFs to evaluate the therapeutic effect on KFs. RESULTS: We discovered that PLK4 was overexpressed in keloid dermal samples and KFs compared with adjacent normal skin samples and NFs derived from the same patients. High PLK4 expression was positively associated with the proliferation, migration, and invasion of KFs. Furthermore, knockdown of PLK4 expression or inhibition of PLK4 activity by Cen-B suppressed KF growth, induced KF apoptosis via the caspase-9/3 pathway, and induced cell cycle arrest at the G0/G1 phase in vitro. CONCLUSIONS: These findings demonstrate that PLK4 is a critical regulator of KF proliferation, migration, and invasion, and thus, Cen-B is a promising candidate drug for keloid treatment.

Long-Term Complications from Diced Cartilage in Rhinoplasty: A Meta-Analysis.

Objective: To analyze the incidences of long-term complications and revision surgery associated with diced cartilage grafts in dorsal augmentation rhinoplasty. Methods: The PubMed, MEDLINE, Embase, and Cochrane Central Register of Controlled Trials databases were searched for clinical studies on the use of diced cartilage for dorsal augmentation published. A meta-analysis was conducted to pool the estimated rates of infection, overcorrection, visible irregularity, absorption, and revision surgery. Result: A total of 14 studies involving 2380 patients were included in the systematic review. The combined rates were 11.5% for overall complications and 5.3% for revision surgery. The rates of the most frequently reported complications were 4.5% for infection, 5.3% for visible irregularity, 0.7% for overcorrection, and 0.5% for absorption. There was no significant difference in the rates of visible irregularity (p = 0.23) and revision surgery (p = 0.71) among the wrapped diced cartilage, glued diced cartilage, and free diced cartilage groups. Conclusion: This meta-analysis presents the first comprehensive and quantitative report of long-term complications associated with diced cartilage in dorsal augmentation rhinoplasty. Infection and visible irregularity were the most frequently reported complications. The rates of irregularity and revision surgery were not correlated with the diced cartilage packing methods.

Autologous fat transfer rescues expanded skin from expansion failure: A retrospective cohort study in Asians.

BACKGROUND: Soft tissue expansion is a common technique for the regeneration of extra skin to repair skin defects. However, some warning signs like skin thinning and telangiectasia are often found during the expansion process, which indicates the skin flaps cannot be further expanded. These signs may result in the suspension of expansion or ultimately jeopardize the final outcome. Fat grafting is used to treat these potential complications and enable the continuation of the expansion procedure in some cases. In this study, we aimed to investigate the efficiency and safety of fat grafting in this process. METHODS: The study was conducted on patients from January 2012 to December 2017 with warning signs of expansion treated with fat grafting (treatment group) or pause expansion (control group). Follow-up data, such as expansion status, dermal thickness, telangiectasia, skin texture using volume assessment, B-mode ultrasound, and semiquantitative scoring, were collected. RESULTS: A total of 67 expanded skin regions with warning signs were enrolled. The expansion fold increased 2.14-fold at 12 weeks after treatment compared with 0.74-fold in control (P=0.02). The semiquantitative score was significant improved at 4 weeks (9.03 ± 0.73 vs. 7.45 ± 0.55; p=0.033). Meanwhile, the skin thickness in the experimental group did not show decreasing trend even in the continued expansion process. CONCLUSIONS: Autologous fat grafting represents an effective and safe method to rescue expanded skin from limited skin regeneration. This technique also represents a valuable tool to increase the chances for further expansion.

Perioperative Care of Children with Acute Exogenous Lipoid Pneumonia Submitted to BAL/FB.

OBJECTIVE: The present study evaluates the psychosocial care and the bronchoalveolar lavage (BAL)/fiberoptic bronchoscopy (FB) procedure in children with pediatric acute exogenous lipoid pneumonia (ELP) and summarizes the critical points of nursing. METHODS: Data on the psychosocial factors of the patients and clinical information were collected. Participants comprised 41 children within three years of age. RESULTS: All the children were cooperative with the BAL/FB procedure. The children's pain scores were between 4-6, and the psychological conditions of the children and caregivers were nervous/anxious upon admission. After the medical staff's psychological care and health education, the children's postoperative pain scores were reduced to 0-3, and the psychological state of the caregivers was positive. CONCLUSION: Psychological care can alleviate families' adverse emotions and promote treatment cooperation and recovery from the acute ELP.

Sequential Enzymatic Digestion of Different Cartilage Tissues: A Rapid and High-Efficiency Protocol for Chondrocyte Isolation, and Its Application in Cartilage Tissue Engineering.

OBJECTIVE: The classic chondrocyte isolation protocol is a 1-step enzymatic digestion protocol in which cartilage samples are digested in collagenase solution for a single, long period. However, this method usually results in incomplete cartilage dissociation and low chondrocyte quality. In this study, we aimed to develop a rapid, high-efficiency, and flexible chondrocyte isolation protocol for cartilage tissue engineering. DESIGN: Cartilage tissues harvested from rabbit ear, rib, septum, and articulation were minced and subjected to enzymatic digestion using the classic protocol or the newly developed sequential protocol. In the classic protocol, cartilage fragments were subjected to one 12-hour digestion. In the sequential protocol, cartilage fragments were sequentially subjected to 2-hour first digestion, followed by two 3-hour digestions. The collected cells were then subjected to analyses of cell-yield efficiency, viability, proliferation, phenotype, and cartilage matrix synthesis capacity. RESULTS: Overall, the sequential protocol exhibited higher cell-yield efficiency than the classic protocol for the 4 cartilage types. The cells harvested from the second and third digestions demonstrated higher cell viability, more proliferative activity, a better chondrocyte phenotype, and a higher cartilage-specific matrix synthesis ability than those harvested from the first digestion and after the classic 1-step protocol. CONCLUSIONS: The sequential protocol is a rapid, flexible, high-efficiency chondrocyte isolation protocol for different cartilage tissues. We recommend using this protocol for chondrocyte isolation, and in particular, the cells obtained after the subsequent 3-hour sequential digestions should be used for chondrocyte-based therapy.

A randomized, controlled clinical trial of autologous stromal vascular fraction cells transplantation to promote mechanical stretch-induced skin regeneration.

BACKGROUND: The regeneration response of the skin to mechanical stretching in vivo has been explored in reconstructive surgery to repair large-scale deformities. The ability of the skin to regenerate limits the reconstructive outcome. Here, we propose an approach in which autologous stromal vascular fraction (SVF) cells and mechanical stretching are combined to overcome this limitation and promote skin regeneration. METHODS: This randomized, blinded, placebo-controlled clinical trial screened 22 participants undergoing tissue expansion with exhausted regeneration. Twenty eligible participants received intradermal injections of the SVF or placebo treatments. Follow-ups were conducted at 4, 8, and 12 weeks to assess efficacy and at 2 years to assess safety. The primary endpoint was the expanded skin thickness at 12 weeks. The secondary endpoints included skin thickness at 4 and 8 weeks, the expansion index (EI), and the skin texture score at 12 weeks. RESULTS: The skin thickness of the SVF group was significantly higher than that of the control group at both 8 weeks (mean difference 0.78 [95% CI - 1.43 to - 0.11]; p = 0.018) and 12 weeks (0.65 [95% CI - 1.30 to - 0.01]; p = 0.046). In the SVF group, the increase in skin thickness was significant at 4 weeks (0.49 [95% CI - 0.80 to - 0.06]; p = 0.010) to 8 weeks (0.45 [95% CI - 0.92 to 0.02]; p = 0.026) and maintained after 12 weeks, whereas that in the control group was reduced after 8 weeks (0.42 [95% CI - 0.07 to 0.91]; p = 0.037). The SVF group showed greater EI increases than the control group (0.50 [95% CI - 0.00 to 0.99]; p = 0.047). The skin texture scores in the SVF group were greater than those in the control group at 12 weeks. Histologically, SVF-treated expanded skin showed more proliferating cells and blood vessels, and the extracellular matrix volume increased. No severe adverse events occurred. CONCLUSIONS: Transplantation of SVF cells can expedite the potency of mechanical stretch-induced skin regeneration and provide clinical reconstruction with plentiful tissue. TRIAL REGISTRATION: This trial was registered with the Chinese Clinical Trial Registry, ChiCTR2000039317 (registered 23 October 2020-retrospectively registered).