Autologous Cell Suspension Research Articles

A novel method for the establishment of autologous skin cell suspensions: characterisation of cellular sub-populations, epidermal stem cell content and wound response-enhancing biological properties.

Introduction: Autologous cell suspension (ACS)-based therapy represents a highly promising approach for burns and chronic wounds. However, existing technologies have not achieved the desired clinical success due to several limitations. To overcome practical and cost-associated obstacles of existing ACS methods, we have established a novel methodology for rapid, enzymatic disaggregation of human skin cells and their isolation using a procedure that requires no specialist laboratory instrumentation and is performed at room temperature. Methods: Cells were isolated using enzymatic disaggregation of split-thickness human skin followed by several filtration steps for isolation of cell populations, and cell viability was determined. Individual population recovery was confirmed in appropriate culture medium types, and the presence of epidermal stem cells (EpSCs) within keratinocyte sub-populations was defined by flow cytometry via detection of CD49 and CD71. Positive mediators of wound healing secreted by ACS-derived cultures established on a collagen-based wound-bed mimic were detected by proteome arrays and quantified by ELISA, and the role of such mediators was determined by cell proliferation assays. The effect of ACS-derived conditioned-medium on myofibroblasts was investigated using an in-vitro model of myofibroblast differentiation via detection of α-SMA using immunoblotting and immunofluorescence microscopy. Results: Our methodology permitted efficient recovery of keratinocytes, fibroblasts and melanocytes, which remained viable upon long-term culture. ACS-derivatives comprised sub-populations with the CD49-high/CD71-low expression profile known to demarcate EpSCs. Via secretion of mitogenic factors and wound healing-enhancing mediators, the ACS secretome accelerated keratinocyte proliferation and markedly curtailed cytodifferentiation of myofibroblasts, the latter being key mediators of fibrosis and scarring. Discussion: The systematic characterisation of the cell types within our ACS isolates provided evidence for their superior cell viability and the presence of EpSCs that are critical drivers of wound healing. We defined the biological properties of ACS-derived keratinocytes, which include ability to secrete positive mediators of wound healing as well as suppression of myofibroblast cytodifferentiation. Thus, our study provides several lines of evidence that the established ACS isolates comprise highly-viable cell populations which can physically support wound healing and possess biological properties that have the potential to enhance not only the speed but also the quality of wound healing.

518 Autologous Skin Cell Suspensions for Reconstruction of Nonthermal Soft Tissue Wounds: A Case Series

Abstract Introduction Based on studies showing the use of autologous skin cell suspensions (ASCS) leads to burn wound closure with significantly smaller amounts of harvested donor skin, the Food and Drug Administration approved the first ASCS for the reconstruction of thermal injuries in September, 2018. The benefit of needing less donor skin to heal a given soft tissue wound has led to off-label usage of ASCS in a variety of clinical situations, such as necrotizing soft tissue infections (NSTI), toxic epidermal necrolysis (TEN), and traumatic injuries with soft tissue loss among others. Here, we report on a single-institution case series of ASCS usage for closure of soft tissue wounds caused by nonthermal mechanisms. Methods We performed a retrospective chart review of all patients treated with ASCS for an indication of non-thermal soft tissue wounds at our American Burn Association-verified academic burn center between April 1, 2018 to June 1, 2022. Descriptive demographic data were collected. Results Eight patients (6 male/2 female, mean age 57 + 17 yrs, mean length of stay 46 + 30 days) were found to have undergone closure of their wounds using ASCS during the time period of the chart review. Indications were NSTI (n=2), frostbite (n=2), antiphospholipid syndrome (n=2), and TEN (n=2). ASCS was used as a sole therapy for the TEN patients while ASCS was used in combination with meshed autografting for all other patients. The mean size of wounds treated with ASCS was 2835 + 2670 cm2, and the mean ASCS take rate was 86 + 7%. No ASCS-related complications were found. Conclusions These results bolster the off-label use of ASCS solutions for closure of nonthermal wounds while the surgical world awaits the results of an ongoing Phase III trial on this question Applicability of Research to Practice This study seeks to contribute to the literature supporting off-label uses of ASCS in non-thermal injuries & diseases.

559 Verticalization Therapy for ICU-Level Burn Patient: A Case Study

Abstract Introduction The entire interdisciplinary team can be faced with many challenges when treating a bariatric, critically ill patient with significant burn injuries. Some studies suggest that the care of these patients increases staff workload and leads to the need for increased assistance for proper turning, positioning, and completion of wound care. When caring for these types of patients, patient and staff safety should be considered, as well as what will ensure the best patient outcomes. Our rehab team initiated the use of verticalization therapy with a patient on the burn unit with 28% TBSA full-thickness burns to back, chest, BUEs and a BMI of 45.8 in order to promote improved healing, cognitive status, and pulmonary function. While verticalization therapy is described for use in patients with pulmonary issues or invasive cardiac support devices, this was the first time this therapy was used in our busy ABA-verified burn center. Methods Once deemed appropriate for participation with PT/OT, a collaborative protocol with nursing, therapy, and burn provider team was developed. The patient was transitioned into a specialized bed allowing for verticalization up to 90 degrees. The patient was incrementally verticalized by 10 degrees at a time while monitoring vitals, assessing patient safety and pain response. Ultimately, the patient reached 40 degrees vertical and maintained this position for up to 2 hours. This intervention was utilized by both rehab specialists and nursing staff and was taught to nursing staff to be performed daily until patient was able to actively participate and tolerate edge of bed or out of bed activity. Results Use of the verticalization positioning was used to promote weight bearing activity, improve pulmonary function, improve cognitive status through upright positioning, and for pressure relief of graft areas specifically for offloading pressure to back wounds and grafts. The bed was used for 1 month. During this time, the patient successfully liberated from mechanical ventilation and shock state and mobilized with therapy assistance. The patient demonstrated improved strength, active participation with therapies, and decreased agitation as the month progressed. Autograft split-thickness skin grafts with autologous cell suspension epithelial autografts to the patient’s posterior torso healed well and were fully closed by the time of discharge to inpatient rehabilitation. Conclusions Use of verticalization beds can be implemented in the care of patients with burn injuries safely and successfully. Further research regarding skin healing related to use of the bed and verticalization therapy in burn injured patients should be conducted. Applicability of Research to Practice This intervention can be used for patients of all sizes and can be implemented as soon as hospital day one, if the patient is medically appropriate

Cell-based therapy in thin endometrium syndrome

Of the known causes of uterine factor infertility, Ashermans syndrome or the so-called intrauterine synechia, chronic endometritis and endometrial hypoplastic processes are most often distinguished. Thin endometrial syndrome is characterized by a decrease in the thickness of the endometrium to 7 mm or less in the proliferative phase of the menstrual cycle and a multiple decrease in the frequency of embryo implantation. Numerous treatment strategies have so far been proposed for treating refractory thin endometrium syndrome. Recently, cell therapy has been proposed as an ideal alternative for endometrium regeneration, including the employment of stem cells, platelet-rich plasma, and growth factors as therapeutic agents. Single center, prospective, open-label study of efficacy of cell-based therapy in the complex treatment of thin endometrium syndrome in patients with infertility was conducted. The study involved 36 women aged 28 to 36 years, the middle age was 34.2 1.1 years. All patients included in the study received 3 cycles of intrauterine administration of a suspension of autologous stem cells isolated from bone marrow. Bone marrow was successfully aspirated from the iliac crest in all patients. Mononuclear cells was isolated by density gradient centrifugation according to the standard method. The cell material was cryopreservated and thawed immediately before administration. The procedure for intrauterine transplantation of isolated but not cultured cells was performed on the 5th7th day of the menstrual cycle. There were no significant adverse events related to harvest or administration. The thickness of the endometrium before and after treatment was 2.39 0.64 and 6.56 0.94 mm (t = 21.94, p = 0.0001), respectively. The rate of patients with an endometrial thickness of more than 7 mm after treatment was 77.8%. The effectiveness of assisted reproductive technology in patients with normal endometrial (n = 28) was 32.1%. Immunohistochemistry confirms the presence of chronic endometritis before and after treatment in 22 (61.1%) and 19 (52.8%) patients, respectively, while after treatment a significant decrease in the levels inflammatory markers was found.

601 Bromelain Based Enzymatic Debridement Followed by Application of Autologous Cell Suspension for Treatment of Burns

IntroductionBromelain based enzymatic debridement has been shown to have a unique selectivity for nonviable tissue and is being evaluated for its use in the treatment of select burn wounds. The treatment has potential to augment more traditional operative eschar excision. Herein, we retrospectively review our tertiary burn center’s early experiences in cases in which bromelain based enzymatic debridement (BBED) is paired with autologous cell suspension (ACS).MethodsPatients whose burn wounds were deemed eligible for BBED at our institution from July 2020 to June 2021 were queried for concurrent treatment with ACS. Inclusion criteria by study design consisted of 18 years of age or older, non-electrical and non-chemical burns, predominance of deep partial and/or full thickness burn, and less than 30% total body surface area. Success of escharotomy by BBED, grafting methods, infections, transfusions, inflammatory response, and response to grafting were analyzed.ResultsSeven patients met inclusions criteria from July 2020 to June 2021. The mean age was 44 (IQR: 39-53). The mean TBSA was 13.7% (IQR 8-20). The mean time from injury to BBED was 2.1 days (IQR 1.5-2.0). The mean time to ACS after BBED was 1.3 days (IQR 1.0-1.5). The mean area debrided with BBED was 2347 sq cm (IQR 1367-3534). All patients were determined to have complete eschar removal on the day of application by the multidisciplinary burn team. All seven cases had deep partial thickness areas treated with ACS alone, with a mean area of 1574sq cm (IQR 877-2327). The total area treated was 11,016 sq cm. Only two patients required delayed grafting, a combined total of 1573 sq cm, meaning that 86% of the total burn area treated with ACS alone was recovered. Five patients had ACS paired with split thickness autografts, with a mean area of 867 sq cm (IQR 519-1328). Graft imbibition was noted to be rapid with >95% wound closure by postoperative day eight. There were no graft infections, and no transfusions required. White blood cell (WBC) counts decreased after BBED, then increased marginally after grafting. Only two patients had WBC >20,000 and this aided in diagnosis of one donor site infection and one pneumonia.ConclusionsIn our series, BBED within three days of injury paired with ACS within two days of BBED resulted in excellent split thickness graft take and recovery of 86% of the burn wounds treated with ACS alone.

804 Catastrophic Burn Management: A case series using autologous skin cell suspension

IntroductionCatastrophic thermal injury treatment is complex due to the lack of autologous donor site, which is imperative for permanent wound closure. Historically, our burn unit has relied on application of cultured epithelial autografts for wound closure in this patient population. Lab grown skin requires a significant time investment. Therefore, in our most recent subset of large burn injuries, we have transitioned to the use of autologous skin cell suspension over widely meshed autograft utilization almost exclusively.MethodsA case series of four thermally injured patients with injuries greater than 75% TBSA was reviewed. Patient length of stay, operative interventions, skin coverage, mortality and cost were examined.ResultsBurn size ranged from 75-85% TBSA in four patients. Early excision was implemented for all four patients in the case study. One of the four patients was treated with a combination of lab grown skin and autologous skin cell suspension. The three other patients were treated exclusively with autologous skin cell suspension for wound coverage. All patients underwent widely meshed autograft with the autologous cell suspension for wound coverage. All four patients survived to discharge and their initial follow up. Cost of operative interventions were less expensive for autologous skin cell suspension than for cultured epithelial autografts. Length of stay varied from 66-179 days. Discharge location varied from acute rehabilitation, to a homeless shelter, to another burn facility with inpatient psychiatric services.ConclusionsAutologous skin cell suspension is a safe and efficient treatment modality to obtain permanent wound closure in catastrophic thermal injuries.

603 Timing of autologous spray cell suspension: better early than late

IntroductionAutologous spray cell suspension is associated with multiple beneficial outcomes, however, the optimal timing of use has not been determined. We examined the timing of spay cell application, and hypothesize that early (< 72 hr) use is associated with faster definitive wound coverage, impacting multiple clinical outcomes compared to late ( >72 hr) use of spray cells.MethodsA retrospective review of 28 patients with spray cell application from March 2020 -- May 2021 was conducted. Pediatric patients < 16 yrs (n=1) as well as deaths (n=2--including 1 withdrawal) were excluded, leaving 25 patients. Those that received spray cells early (< 72 hrs, n=14) were compared to late ( >72 hrs, n=11). Time to index operation, time to spray cell application, rate of complete wound coverage, number of OR trips required for wound closure, wound infection rates, LOS / %TBSA, and overall hospital LOS were examined.ResultsThere was no difference in demographics or % TBSA burn (21.4% vs. 23/5%), between early and late spray cell groups. The early group had a significantly faster time to index operation (1.2 dys vs. 2.5 dys P= .05), and faster time to spray cell application (1.6 dys vs. 18.1 dys P=.036) (Table 1). The early group had a greater wound coverage rate at index operation —64.2% complete coverage vs. 18.1% in the late group (p< .03). The total number of OR trips needed for wound coverage was less in the early group (1.5 trips / pt) vs. the late (7.0 trips / pt) P=0.03. The early group had less wound infections, a shorter LOS / % TBSA burn ( 0.83 dys vs. 1.85 dys P=0.005), and shorter overall LOS (16.5 dys vs. 46.9 dys P=0.03).ConclusionsAutologous spray cell use has multiple beneficial outcomes, however, the optimal timing of application has not been determined. We report that early (< 72 hr) application of spray cells is associated with greater rate of complete wound coverage at index operation, decreased total number of operations needed for wound coverage, less wound infections, shorter LOS / %TBSA, and shorter overall LOS. This is likely related to earlier definitive wound coverage and closure, which is afforded by the large expansion ratio of spray cells.

Evaluation of healing outcomes combining a novel polymer formulation with autologous skin cell suspension to treat deep partial and full thickness wounds in a porcine model: a pilot study

Autologous skin cell suspensions (ASCS) can treat burns of varying depths with the advantage of reduced donor site wound burden. The current standard primary dressing for ASCS is a nonabsorbant, non-adherent, perforated film (control) which has limited conformability over heterogeneous wound beds and allows for run-off of the ASCS. To address these concerns, a novel spray-on polymer formulation was tested as a potential primary dressing in porcine deep partial thickness (DPT) and full thickness (FT) wounds. It was hypothesized that the polymer would perform as well as control dressing when evaluating wound healing and scarring.DPT or FT wounds were treated with either a spray-on poly(lactic-co-glycolic acid) (PLGA) and poly(lactide-co-caprolactone) (PLCL) formulation or control ASCS dressings. Throughout the experimental time course (to day 50), we found no significant differences between polymer and control wounds in % re-epithelialization, graft-loss, epidermal or dermal thickness, or % dermal cellularity in either model. Pigmentation, erythema, elasticity, and trans-epidermal water loss (TEWL), were not significantly altered between the treatment groups, but differences between healing wounds/scars and un-injured skin were observed. No cytotoxic effect was observed in ASCS incubated with the PLGA and PLCL polymers.These data suggest that the novel spray-on polymer is a viable option as a primary dressing, with improved ease of application and conformation to irregular wounds. Polymer formulation and application technique should be a subject of future research.

Experimental assessment and comparative analysis of efficacy for modified method of cellular therapy in chronic wounds on background of diabetes mellitus

Objective. Determination of efficacy of the cellular therapy modified method in chronic wounds on background of diabetes mellitus, comparing with standard and new methods of treatment in experiment.&#x0D; Materials and methods. Experimental investigations were conducted on 30 adult male rats of a Wistar line, in which diabetes mellitus was experimentally simulated. Full-thickness square wound was used as experimental pattern. In the animals of group A (control) the wounds were processed with 0.05% solution of chlorhexidine bigluconate and the hydrohel bandages put on (standard of treatment). To the group B animals photodynamic therapy was conducted, and processes of regeneration were stimulated with the help of the mesenchymal stem cells application. To the group C animals the wounds bottom and edges were infiltrated with suspension of autologous mesenchymal stem cells for active regeneration after conduction of photodynamic therapy.&#x0D; Results. Best healing of the wounds while application of photodynamic therapy and injections of mesenchymal stem cells was noted: on the 5th day from the treatment beginning the wounds square reduction have appeared the most – 71.9%, while application of mesenchymal stem cells this index have constituted 61.6%, and while standard treatment – 36.0%.&#x0D; Conclusion. Treatment of chronic wounds on background of diabetes mellitus, using photodynamic therapy and injections of mesenchymal stem cells, have demonstrated essentially better results, than after standard method. Application of mesenchymal stem cells into the wound, the results of which were compared with results of their injectional introduction, may be recommended as alternative, if immediate injections of mesenchymal stem cells into the tissues over the wound are impossible.

Efficacy of stromal vascular fraction and enzyme-free mechanical isolation therapy in experimental full thickness burn wounds

Background Autologous cell suspensions obtained by a stromal vascular fraction (SVF) and enzyme-free mechanical isolation (EMI) are an alternative in the treatment of burn wounds. In this study, we aimed to investigate the effect of autologous cell suspensions obtained by SVF and EMI on full-thickness skin burn wounds. Methods A total of 45 male Sprague–Dawley rats were divided into three groups, SVF group, EMI group, and SVF + EMI group. The groups were also classified as the first, second, and third week of the burn to reveal the effect of the treatment on the burn in the early, middle, and late stages. For treatment, 0.2 ml SVF or 0.2 ml EMI was injected subcutaneously into the burn lesions of the subjects. Histopathological examination was performed on the burn wounds taken at the end of the experiment, and Ki67, CD44, CD73, CD90, and CK17 expressions were evaluated. Results Histological examination revealed that there was no improvement in the control samples, but the skin was multicellular, vascularization was present. Histologic scores in all groups was significantly better than control, and SVF + EMI was the best group in terms of recovery (p < 0.05). Ki67, CK17, CD44, CD73, and CD90 expressions were significantly higher in the treatment groups compared to the control (p < 0.05). Conclusion We found in our study that both applications significantly increased the healing of the burn wound. Moreover, SVF + EMI application provided more improvement than SVF or EMI alone.

Применение регенеративных клеточных технологий при лечении тяжёлых местных лучевых поражений у пострадавших в чрезвычайных ситуациях: из опыта работы специалистов Федерального медицинского биофизического центра им. А.И.Бурназяна ФМБА России

It has been noted that one of the most common types of radiation injuries when a person is exposed to ionizing radiation is radiation burns — severe local radiation injuries. The aim of the study is to apply stromal and vascular fraction of adipose tissue to increase the efficiency of complex therapy for local radiation lesions. Materials and methods of the study. In 2017-2019, 7 patients (all male; mean age — (54.83±9.41) years) with local radiation lesions of the skin — ulcerative-necrotic lesions of the skin and underlying tissues — were treated at the Federal Medical Biophysical Center named after A.I. Burnazyan of FMBA of Russia. For more than 6 months the patients received conventional conservative therapy of local radiation lesions and a single injection of cell suspension of autologous cells of stromal-vascular fraction of adipose tissue — the average number of cells was (60.33×106±64.04). Results of the study and their analysis. All patients had no serious adverse events and reactions associated with the introduction of autologous regenerative cells of adipose tissue. During the whole period of observation after stromal-vascular fraction of adipose tissue was injected, late radiation ulcers remained without signs of inflammation and infiltration. The patients were discharged from the hospital in satisfactory condition. According to the authors, the use of stromal-vascular fraction of adipose tissue in local radiation lesions provides favorable conditions: to increase the effectiveness of complex therapy; to reduce healing time of the wound surface; to regulate and activate immune and reparative processes in the dermis; to restore the damaged vascular network, lost skin without severe scarring changes; to heal and achieve a satisfactory result, decent quality of life of patients.

A Pilot Study of Negative Pressure Therapy with Autologous Skin Cell Suspensions in a Porcine Model

Negative pressure wound therapy (NPWT) is an option for securing meshed split thickness skin grafts (mSTSGs) after burn excision to optimize skin graft adherence. Recently, the use of autologous skin cell suspension (ASCS) has been approved for use in the treatment of burn injuries in conjunction with mSTSGs.To date, limited data exists regarding the impact of NPWT on healing outcomes when the cellular suspension is utilized. It was hypothesized that NPWT would not negatively impact wound healing of ASCS+mSTSG. A burn, excision, mSTSG, ASCS ± NPWT model was used. Two Duroc pigs were utilized in this experiment, each with 2 sets of paired burns. Four wounds received mSTSG+ASCS+NPWT through post-operative day 3, and 4 wounds received mSTSG+ACSC+ traditional ASCS dressings. Cellular viability was characterized prior to spraying. Percent re-epithelialization, graft-adherence, pigmentation, elasticity, and blood perfusion and blood vessel density were assessed at multiple time points through 2 weeks. All wounds healed within 14 days with minimal scar pathology and no significant differences in percent re-epithelialization between NPWT, and non-NPWT wounds were observed. Additionally, no differences were detected for pigmentation, perfusion, or blood vessel density. NPWT treated wounds had less graft loss and improved elasticity, with elasticity being statistically different. These data suggest the positive attributes of the cellular suspension delivered are retained following the application of negative pressure. Re-epithelialization, revascularization, and repigmentation are not adversely impacted. The use of NPWT may be considered as an option when using ASCS with mSTSGs for the treatment of full-thickness burns.

Outcomes for Hand Burns Treated With Autologous Skin Cell Suspension in 20% TBSA and Smaller Injuries.

In order to address the confounder of TBSA on burn outcomes, we sought to analyze our experience with the use of autologous skin cell suspensions (ASCS) in a cohort of subjects with hand burns whose TBSA totaled 20% or less. We hypothesized that the use of ASCS in conjunction with 2:1 meshed autograft for the treatment of hand burn injuries would provide comparable outcomes to hand burns treated with sheet or minimally meshed autograft alone. A retrospective review was conducted for all deep partial and full-thickness hand burns treated with split-thickness autograft (STAG) at our urban verified burn center between April 2018 and September 2020. The exclusion criterion was a TBSA greater than 20%. The cohorts were those subjects treated with ASCS in combination with STAG (ASCS(+)) vs those treated with STAG alone (ASCS(-)). All ASCS(+) subjects were treated with 2:1 meshed STAG and ASCS overspray while all ASCS(-) subjects had 1:1, piecrust, or unmeshed sheet graft alone. Outcomes measured included demographics, time to wound closure, proportion returning to work (RTW), and length of time to RTW. Mann-Whitney U test was used for comparisons of continuous variables and Fisher's exact test for categorical variables. Values are reported as medians and 25th and 75th interquartile ranges. Fifty-one subjects fit the study criteria (ASCS(+) n = 31, ASCS(-) n = 20). The ASCS(+) group was significantly older than the ASCS(-) cohort (44 [32-54] vs 32 years [27.5-37], P = .009) with larger %TBSA burns (15% [9.5-17] vs 2% [1-4], P < .0001) and larger size hand burns (190 [120-349.5] vs 126 cm2 [73.5-182], P = .015). Comparable results were seen between ASCS(+) and ASCS(-), respectively, for time to wound closure (9 [7-13] vs 11.5 days [6.75-14], P = .63), proportion RTW (61% vs 70%, P = .56), and days for RTW among those returning (35 [28.5-57] vs 33 [20.25-59], P = .52). The ASCS(+) group had two graft infections with no reoperations, while ASCS(-) had one infection with one reoperation. No subjects in either group had a dermal substitute placed. Despite being significantly older, having larger hand wounds, and larger overall wounds within the parameters of the study criteria, patients with 20% TBSA burns or smaller whose hand burns were treated with 2:1 mesh and ASCS overspray had comparable time to wound closure, proportion of RTW, and time to return to work as subjects treated with 1:1 or piecrust meshed STAG. Our group plans to follow this work with scar assessments for a more granular picture of pliability and reconstructive needs.

Initial Experience Combining Negative Pressure Wound Therapy With Autologous Skin Cell Suspension and Meshed Autografts.

The success of autologous split-thickness skin grafts (STSGs) in the treatment of full-thickness burns is often dependent on the dressing used to secure it. Tie-over bolsters have been used traditionally; however, they can be uncomfortable for patients and preclude grafting large areas in one definitive operation. Negative pressure wound therapy (NPWT) is used as an alternative to bolster dressings and may afford additional wound healing benefits. In our center, NPWT has become the dressing of choice for securing STSGs. While the RECELL® system is being used in conjunction with STSGs, it is currently unknown whether autologous skin cell suspensions (ASCS) can be used with NPWT. This report is a retrospective chart review of nine patients treated in this manner. All wounds were almost completely re-epithelialized within 14 days, and their healing was as expected. Wound healing trajectories are shown. There were no significant complications in these patients. This dressing technique can be considered as an option when using ASCS and widely meshed STSG.

638 Biodegradable Temporizing Matrix in Combination with Wide Meshed Graft and Autologous Spray Cell Suspension (ASCS) Graft Can be Used for Reconstruction of Extensive Necrotizing Infections: A Case Report

Abstract Introduction Background: A necrotizing infection of the left upper extremity underwent extensive debridement with remaining exposed muscle, tendon, and bone. BTM was used to provide an acceptable temporizing matrix in conjunction with wide meshed split thickness skin graft (STSG) 3:1 ratio and ASCS graft for successful reconstruction. This left a functional limb and avoided arm forequarter amputation. Methods Case Presentation: We present a 67 y/o male with necrotizing infection who underwent extensive surgical debridement of skin and subcutaneous tissues of the left hand, forearm, and upper arm. BTM was applied for coverage over muscle, tendon, and bone to salvage his arm and avoid forequarter amputation. Following maturation of the BTM a 3:1 ratio STSG was placed along with application of an ASCS graft. A vacuum assisted closure (VAC) dressing was successfully used to stabilize the grafts. One month post grafting the wound was approximately 94% healed, with good range of motion, and limited but improving function of his arm. Results Conclusion: Necrotizing infection extremity reconstruction can be achieved with BTM, wide meshed STSG, and autologous ASCS grafting. A wound VAC provided a safe and effective dressing over these grafted mediums. Conclusions Conclusion: Necrotizing infection extremity reconstruction can be achieved with BTM, wide meshed STSG, and ASCS epidermal autograft. A wound VAC provided a safe and effective dressing over these grafted mediums.

Rat epidermal stem cells promote the angiogenesis of full-thickness wounds

BackgroundFull-thickness wounds severely affect patients’ life quality and become challenging problems for clinicians. Stem cells have great prospects in the treatment of wounds. Our previous study confirmed that autologous basal cell suspension could promote wound healing, and epidermal stem cells (ESCs) were detected in the basal cell suspension. Herein, this study aimed to explore the effect of ESCs on full-thickness wounds.MethodsRat ESCs were isolated and expanded and then were transfected with lentivirus to stably express enhanced green fluorescent protein. The experimental rats were randomly divided into 2 groups: in the ESC group, the rat ESCs were sprayed on the full-thickness wounds of rats; in the control group, phosphate-buffered saline was sprayed the on the wounds. Next, wound healing and neovascularization were evaluated. Colonization, division, and differentiation of ESCs on the wound were analyzed by immunofluorescence.ResultsThe rat ESCs colonized, divided, and proliferated in the wound. Additionally, rat ESCs around blood vessels differentiated into vascular endothelial cells and formed a lumen-like structure. Compared with the control group, the ESC group showed enhanced angiogenesis and accelerated wound healing.ConclusionsOur study confirmed that rat ESCs are safe and effective for treating full-thickness wounds. Additionally, under certain conditions, ESCs can differentiate into vascular endothelial cells to promote angiogenesis and wound healing.

727 Use of a Biodegradable Dermal Matrix in Conjunction with Meshed and Sprayed Autologous Cell Suspension with a Vacuum Assisted Dressing Achieved Limb Salvage in a 73 Year Old with Deep Full Thickness Burns Despite Exposed Tendon and Bone

Abstract Introduction Deep Full thickness burns require tangential excision of the burned tissues down to viable tissue. This often leaves exposed muscle, tendon and bone. Coverage of these areas is vital to limb salvage. Multiple modalities are necessary with these type of patients, such as, dermal matrix graft, split thickness skin graft, sprayed autologous cell suspension (ACS) and wound vacuum therapy(VAC). A dermal matrix provides a scaffold for tissue and vascular en growth that is suitable for accepting a skin graft and also provides a layer between the muscle, tendon, bone and the skin graft which improves movement and produces a more pliable graft. Biodegradable temporizing matrix(BTM), produced by PolyNovo Ltd, was used to cover the muscle, bone and tendon. This product has a silicone backing which can be delaminated once it has matured with tissue and vascular en growth for auto grafting. A combination of a wide meshed skin graft at 3:1 ratio and a sprayed ACS allows for faster healing and less donor site usage which decreases the overall wound burden. Methods A 73 y/o male with total body surface area burn of 24 % suffered deep full thickness burns to the bilateral lower legs. He underwent multiple debridements with management of the right leg with irrigating wound vacuum therapy. The deeper right leg had a wound base with that consisted of muscle from the thigh down onto the foot with exposed tendons and anterior tibial bone. The open wounds to the right leg were grafted with BTM for dermal substitute coverage. Once the BTM was mature it was delaminated, and the wound surface was covered with a 3:1 ratio split thickness skin graft (STSG) and sprayed on autologous cell suspension. This resulted in using less donor skin than a 1.5 or 2:1 meshed graft STSG which decreased his wound burden. Donor sites were also sprayed with ACS. Results We had nearly 100% take of the STSG and sprayed autologous suspension epidermal graft. There was a small area over the patella tendon where the BTM did not incorporate. This is though to be because of lack of immobilization of his knee and a pseudomonal infection. Donor sites healed faster and there were minimal open areas to the graft POD 11 excluding the patella area. Conclusions BTM can be used to provide a suitable dermal substitute in complex wounds when muscle, tendon and/or bone are exposed. Immobilization of the joints is necessary to allow the tissue en growth into the dermal matrix. Wide meshed graft with sprayed ACS can be used with good results with wound vacuum therapy. Decreased wound burden and time to heal open wounds of the graft and donor sites. Applicability of Research to Practice Use of a dermal matrix, wide mesh split thickness graft and sprayed epidermal ACS with wound vacuum therapy is a good option for limb salvage when vital structures are exposed.

A Comparative Study between Follicular Unit Transplantation and Autologous Non-cultured Non-trypsinized Epidermal Cells Grafting (Jodhpur Technique) in Stable Vitiligo.

Introduction:Several modalities are currently available for the treatment of vitiligo, but generally they do not result in complete cure of the disease. Despite the limitations and some side effects, surgical modalities appear to be the method of choice in recalcitrant stable vitiligo. Both Jodhpur technique (JT) and follicular unit transplantation (FUT) are simpler and cheaper methods, requiring minimal infrastructure.Materials and Methods:Hundred patches in 30 patients were divided into Group A and Group B, that is, hair follicle transplantation and autologous non-cultured non-trypsinized epidermal cell suspension grafting (JT), respectively. Fifty patches were included in each group. Efficacy of both the techniques, extent of pigmentation, and color match assessment were the main objectives.Results:Excellent repigmentation (>75%), at the end of 20 weeks post surgery, was observed in 70% lesions in FUT group and 72% of lesions in JT group. Good response (extent of repigmentation of 50%–75%) was seen in 18% of lesions in FUT group as compared to 26% in JT group. JT and FUT technique were almost equal in producing excellent repigmentation. Color match was same for both the groups, that is, 49 of 50 patches showed same color as surrounding in both groups at the end of 20 weeks. Repigmentation of the depigmented hairs occurred in 11 of 46 patients with associated leukotrichia.Conclusion:Our study indicates that both JT and FUT are safe and effective techniques in terms of repigmentation but JT is somewhat superior to FUT in producing excellent and good repigmentation, side effects, patient satisfaction, and dermatology life quality index reduction, while the color match was almost same with both techniques.

An in vitro feasibility investigation considering primary human melanocytes for spray- grafting of freshly isolated autologous skin cells for burn treatment and a clinical case report

A skin cell-spray grafting technique that enables the on-site application of freshly isolated autologous single cell suspensions was already applied in many cases on caucasian patients with low skin coloration. Our project hypothesis is that these suspensions contain keratinocytes and vital melanocytes, that are of particular interest for the treatment of patients of darker skin color. To test this, we applied an in vitro model, wherein the feasibility of i) isolating and ii) spraying of freshly isolated autologous melanocyte-keratinocyte cell suspensions was investigated. Primary human epidermal keratinocytes (HEKs) and melanocytes (MCs) were isolated from skin biopsies (n=8). Biochemical parameter, cell counts, cell morphology, growth behavior and immunofluorescence results were compared in two groups using MC cultures and co-cultures of MCs with HEKs. Case information on using the method clinically with one patient is included. The sprayed mixed cell suspensions proliferated in all groups without measurable loss of viability, and MCs exhibited a regular cell morphology in monoculture up to passage 4°. The sprayed MCs and HEKs demonstrated in vitro glucose and lactate metabolism that was comparable to the pipetted controls. In co-culture, well distributed CK14+ HEKs and NKI/beteb+ MCs could be demonstrated, which interacted in the in vitro model. The ratio of HEKs : MCs in our primary cultures were microscopically counted (n=8 each) as mean +/- SD 1,211,000 (+/- 574,343) HEK : 99,625 (+/- 59,025) MC; i.e., a ratio of approx. 12 : 1. Using the isolation method clinically for a patient with dark skin coloration after suffering severe second-degree burns shows a satisfying re-pigmentation of the resulting wound post healing. Freshly isolated spray-on melanocyte/keratinocyte suspensions provide for a considerable amount of viable HEKs and MCs. Using MCs in spray-grafting suspensions could represent a promising approach for treating severe partial-thickness burns and innovative therapy developments that also aim to address cosmetic aspects.

77 Initial Experience with Autologous Cell Suspension for Treatment of Partial Thickness Facial Burns

77 Initial Experience with Autologous Cell Suspension for Treatment of Partial Thickness Facial Burns