Achieving self-sufficiency in skin allograft: A Singapore experience.

AIM: This paper describes how Singapore achieved skin allograft self-sufficiency in 2017 by adopting 5 key strategies in 2012. BACKGROUND: Singapore General Hospital (SGH) established its own allograft recovery programme in 1998 but was still dependent on overseas allograft procurement. KEY STRATEGIES: RESULTS: The allograft recovery programme expanded from 4 to all 20 institutions. Donor referrals increased by 42.9% from 35 in 2014 to over 50 currently. Donor numbers increased by 210%, rising from 4.5 per year before 2015 to an average of 14 per year from 2015 to 2022. The total allografts recovered increased by 223%, climbing from 13,000 to 42,000 annually. Cryopreservation was adopted, extending shelf life to 5.5 years and doubling storage capacity to more than 140,000 cm2 in 2022. CONCLUSION: Singapore achieved skin allograft self-sufficiency with no overseas procurement since 2017.

Human Umbilical Cord Lining-Derived Epithelial Cells: A Potential Source of Non-Native Epithelial Cells That Accelerate Healing in a Porcine Cutaneous Wound Model.

Human umbilical cord lining epithelial cells [CLECs) are naïve in nature and can be ethically recovered from cords that are routinely discarded. The success of using oral mucosal epithelial cells for cornea defects hints at the feasibility of treating cutaneous wounds using non-native CLECs. Herein, we characterized CLECs using flow cytometry (FC) and skin organotypic cultures in direct comparison with skin keratinocytes (KCs). This was followed by wound healing study to compare the effects of CLEC application and the traditional use of human skin allografts (HSGs) in a porcine wound model. While CLECs were found to express all the epidermal cell markers probed, the major difference between CLECs and KCs lies in the level of expression (in FC analysis) as well as in the location of expression (of the epithelium in organotypic cultures) of some of the basal cell markers probed. On the pig wounds, CLEC application promoted accelerated healing with no adverse reaction compared to HSG use. Though CLECs, like HSGs, elicited high levels of local and systemic immune responses in the animals during the first week, these effects were tapered off more quickly in the CLEC-treated group. Overall, the in vivo porcine data point to the potential of CLECs as a non-native and safe source of cells to treat cutaneous wounds.

Challenges in the management of extensive aplasia cutis congenita.

Aplasia cutis congenita (ACC) is a rare group of congenital disorders characterised by focal or widespread absence of skin, predominantly affecting the scalp. A Malay female infant was born at 37 weeks with extensive ACC, affecting 37% of total body surface area, including her scalp and trunk. There is no consensus on the management of ACC given the rarity and variable presentation. A multi-disciplinary team comprising neonatologists, paediatric dermatologists, plastic surgeons and medical laboratory scientists at the skin bank, employed a more aggressive surgical approach with the aim of avoiding potentially catastrophic morbidity, including sagittal sinus haemorrhage and brain herniation. Out of several surgical options, the team used a staged artificial dermal matrix (Integra) and cultured epithelial autograft application, followed by regular wound dressing, and eventually allowed the child to achieve complete epithelialisation of her trunk, and most of scalp before she was discharged from hospital.

Intraperitoneally Delivered Umbilical Cord Lining Mesenchymal Stromal Cells Improve Survival and Kidney Function in Murine Lupus via Myeloid Pathway Targeting.

To determine the therapeutic efficacy of human umbilical cord lining mesenchymal stromal cells (CL-MSCs) (US Patent number 9,737,568) in lupus-prone MRL/lpr (Faslpr) mice and elucidate its working mechanisms. A total of 4 doses of (20-25) × 106 cells/kg of CL-MSCs was given to 16-week-old female Faslpr mice by intraperitoneal injection. Three subsequent doses were given on 17 weeks, 18 weeks, and 22 weeks, respectively. Six-week-old Faslpr mice were used as disease pre-onset controls. Mice were monitored for 10 weeks. Mouse kidney function was evaluated by examining complement component 3 (C3) deposition, urinary albumin-to-creatinine ratio (ACR), and lupus nephritis (LN) activity and chronicity. Working mechanisms were elucidated by flow cytometry, Luminex/ELISA (detection of anti-dsDNA and isotype antibodies), and RNA sequencing. CL-MSCs improved mice survival and kidney function by reducing LN activity and chronicity and lymphocyte infiltration over 10 weeks. CL-MSCs also reduced urinary ACR, renal complement C3 deposition, anti-dsDNA, and isotype antibodies that include IgA, IgG1, IgG2a, IgG2b, and IgM. Immune and cytokine profiling demonstrated that CL-MSCs dampened inflammation by suppressing splenic neutrophils and monocytes/macrophages, reducing plasma IL-6, IL-12, and CXCL1 and stabilizing plasma interferon-γ and TNF-α. RNA sequencing further showed that CL-MSCs mediated immunomodulation via concerted action of pro-proinflammatory cytokine-induced chemokines and production of nitric oxide in macrophages. CL-MSCs may provide a novel myeloid (neutrophils and monocytes/macrophages)-targeting therapy for SLE.

Keratin-Alginate Sponges Support Healing of Partial-Thickness Burns.

Deep partial-thickness burns damage most of the dermis and can cause severe pain, scarring, and mortality if left untreated. This study serves to evaluate the effectiveness of crosslinked keratin-alginate composite sponges as dermal substitutes for deep partial-thickness burns. Crosslinked keratin-alginate sponges were tested for the ability to support human dermal fibroblasts in vitro and to support the closure and healing of partial-thickness burn wounds in Sus scrofa pigs. Keratin-alginate composite sponges supported the enhanced proliferation of human dermal fibroblasts compared to alginate-only sponges and exhibited decreased contraction in vitro when compared to keratin only sponges. As dermal substitutes in vivo, the sponges supported the expression of keratin 14, alpha-smooth muscle actin, and collagen IV within wound sites, comparable to collagen sponges. Keratin-alginate composite sponges supported the regeneration of basement membranes in the wounds more than in collagen-treated wounds and non-grafted controls, suggesting the subsequent development of pathological scar tissues may be minimized. Results from this study indicate that crosslinked keratin-alginate sponges are suitable alternative dermal substitutes for clinical applications in wound healing and skin regeneration.

The use of negative-pressure wound therapy over a cultured epithelial autograft for full-thickness wounds secondary to purpura fulminans in an infant.

Purpura fulminans is a serious condition that can result in severe morbidity in the pediatric population. Although autologous skin grafts remain the gold standard for the coverage of partial- to full-thickness wounds, they have several limitations in pediatric patients, including the lack of planar donor sites, the risk of hemodynamic instability, and the limited graft thickness. In Singapore, an in-house skin culture laboratory has been available since 2005 for the use of cultured epithelial autografts (CEAs), especially in burn wounds. However, due to the fragility of CEAs, negative-pressure wound therapy (NPWT) dressings have been rarely used with CEAs. With several modifications, we report a successful case of NPWT applied over a CEA in an infant who sustained 30% total body surface area full-thickness wounds over the anterior abdomen, flank, and upper thigh secondary to purpura fulminans. We also describe the advantages of using NPWT dressing over a CEA, particularly in pediatric patients.

A Review of Skin Banking Guidelines and Standards Worldwide: Towards the Harmonization of Guidelines for Skin Banking in Therapeutic Applications for the Regions under the Asia Pacific Burn Association (APBA).

Currently, there are no harmonized guidelines which govern skin banking in the Asia Pacific region. Therefore, skin banks are either unregulated or rely on their nation's legislation or international accreditation to uphold their quality standards. A new set of skin banking guidelines was developed through a comprehensive review and collation of best international practices for the Asia Pacific Burn Association (APBA) members, from donor screening and testing, to skin recovery, processing, storage and distribution, and quality assurance. National regulatory requirements reviewed include the European directives, Australia's Therapeutic Goods Administration and Singapore's tissue banking standards. Further technical and quality management recommendations are referenced from the American Association of Tissue Banks (AATB), the United States Food and Drug Administration standards and guidance documents, various relevant European guides, Japanese Society of Tissue Transplantation guidelines and the Asia Pacific Association of Surgical Tissue Banking. Adapted mainly from the AATB standards, the new Asia Pacific Burn Association Guidelines for Skin Banking in Therapeutic Applications offer a comprehensive manual, addressing: governance and contracts; staff responsibilities; quality management; facilities, equipment and supplies management; donor consent and testing; and recommendations of good practices pertaining to skin recovery, processing, storage and distribution. Besides complementing current generic regulations, they provide technical specifications of major aspects unaddressed in most legislations. This inaugural set of new regional skin banking guidelines would be a start for regional members of the APBA to adopt, and will hopefully culminate in a set of standards so that, in the long run, skin allografts from this region can be of similar quality, which can simplify import process and facilitate the exchange of allografts between members.

Wound healing properties of magnesium mineralized antimicrobial nanofibre dressings containing chondroitin sulphate - a comparison between blend and core-shell nanofibres.

The development of antimicrobial nanofibre dressings that can protect the injured tissues from commensal pathogens while promoting tissue regeneration finds enormous potential in plastic and reconstructive surgery practices. To achieve this goal, we investigated the effect of chondroitin sulphate on the morphology, mechanical properties, wettability and biocompatibility of polydopamine crosslinked electrospun gelatin nanofibres containing mineralized magnesium. To extend the durability of dressings, we prepared composite dressings containing polycaprolactone (PCL) and gelatin as blend or core-shell nanofibres. Nanofibre blends presented greater tensile strength and stretchability, while core-shell nanofibres displayed superior photoluminescent properties. In a porcine model of cutaneous burn injury, both the blend and core-shell nanofibre dressings displayed improved re-epithelialization, wound closure and clinical outcome in comparison to untreated burns. Histology of the biopsied tissues indicated smooth regeneration and collagen organization of the burns treated with core-shell nanostructures than untreated burns. This study compared the physico-chemical and biological properties of composite nanofibres that are capable of accelerating burn wound healing and possess antimicrobial properties, highlighting their potential as wound dressings and skin substitutes.

Chemically defined and xenogeneic-free culture method for human epidermal keratinocytes on laminin-based matrices.

The basal keratinocyte progenitor cells in cultured epithelial autografts (CEAs) regenerate human epidermis after transplantation, a curative therapy for severe burns and, recently, diseases with epidermal loss, such as junctional epidermolysis bullosa (EB). Although a culturing technique for skin keratinocytes was developed four decades ago, the xenogeneic nature of that conventional CEA culture system restricts its use to the treatment of critical and life-threatening cases, such as severe burns on >30% of total body surface area and EB. In the present protocol, we describe how to implement a defined, xeno-free culture system that supports long-term ex vivo expansion of functional human epidermal keratinocytes. Skin-specific basement membrane proteins called laminins play important roles in the maintenance of phenotypic integrity and in supporting the survival of keratinocytes that are adhered to them. This fully human keratinocyte culture system is 'regulatory friendly' and increases the potential of epithelial cellular therapy, which can be expanded to treat less severe burns and other skin defects, such as chronic diabetic wounds. It takes between 7 and 14 d to obtain an initial culture. Conservatively, a secondary culture from the primary culture can be expanded up to 20-fold within 4-5 d once cells reach confluency.

Isolation and Culture of Hair Follicle Dermal Sheath Mesenchymal Stromal Cells.

To date, little is published on the characterization and therapeutic potential of human mesenchymal stromal cells (MSCs) derived from hair follicle dermal sheath (DS). We present protocols for the isolation and culture of human DS-MSCs starting with the use of a dissecting microscope to separate out dermal sheaths from hair follicles for trypsin digestion. We also present the protocols for the adipogenic, osteogenic, and chondrogenic differentiation of these DS-MSCs as we seek to harness these cells for potential applications in stem cell therapy and tissue engineering.

Biologically relevant laminin as chemically defined and fully human platform for human epidermal keratinocyte culture.

The current expansion of autologous human keratinocytes to resurface severe wound defects still relies on murine feeder layer and calf serum in the cell culture system. Through our characterization efforts of the human skin basement membrane and murine feeder layer 3T3-J2, we identified two biologically relevant recombinant laminins-LN-511 and LN-421- as potential candidates to replace the murine feeder. Herein, we report a completely xeno-free and defined culture system utilizing these laminins which enables robust expansion of adult human skin keratinocytes. We demonstrate that our laminin system is comparable to the 3T3-J2 co-culture system in terms of basal markers' profile, colony-forming efficiency and the ability to form normal stratified epidermal structure in both in vitro and in vivo models. These results show that the proposed system may not only provide safer keratinocyte use in the clinics, but also facilitate the broader use of other cultured human epithelial cells in regenerative medicine.

From skin allograft coverage to allograft-micrograft sandwich method: A retrospective review of severe burn patients who received conjunctive application of cultured epithelial autografts.

A 12-year retrospective review of severe burn patients who received cultured epithelial autografts (CEA) at the Singapore General Hospital Burns Centre from January 2005 to December 2016 was carried out. During this period, two different surgical modalities were employed to manage these burn injuries. In the earlier period, following early excision of the burn wounds, exposed surfaces were covered with a combination of split thickness skin autografts (STSG) and allografts. Surfaces covered with skin allografts were subsequently debrided of the allo-epidermis in about 3 weeks later, exposing the allodermis with granulating tissues for grafting of CEA; a technique known as the Cuono's method. In the later period, allograft-autologous micrograft sandwich technique was used to graft on the early excised burns with subsequent CEA grafting. The former and latter groups represented by STSG/C (n=10) and M/CEA (n=14) respectively, were compared in terms of clinical profiles, outcomes, allograft/CEA usage and total graft cost. No significant differences were found based on mean age and presence of inhalation burns between the two treatment methods However, percentage total body surface area (TBSA) and Revised Baux Score were significantly higher (p<0.05) in the M/CEA group compared to the STSG/C group. Differences in clinical outcomes of mortality and length of hospital stay between the 2 groups were statistically insignificant. The average area amount of skin allografts used per patient in the M/CEA group was significantly lower compared to the STSG/C method group which contributed to lower total average cost of grafts used per % TBSA in the M/CEA method group. This might be attributed to the presence of micrografts which seemed to improve stabilization of the wound bed resulting in less operating procedures and improving CEA take. To conclude, the M/CEA method introduced was able to treat more severe burn patients at lower graft costs without compromising critical clinical outcomes significantly.

Skin tissue engineering advances in severe burns: review and therapeutic applications.

Current advances in basic stem cell research and tissue engineering augur well for the development of improved cultured skin tissue substitutes: a class of products that is still fraught with limitations for clinical use. Although the ability to grow autologous keratinocytes in-vitro from a small skin biopsy into sheets of stratified epithelium (within 3 to 4 weeks) helped alleviate the problem of insufficient donor site for extensive burn, many burn units still have to grapple with insufficient skin allografts which are used as intermediate wound coverage after burn excision. Alternatives offered by tissue-engineered skin dermal replacements to meet emergency demand have been used fairly successfully. Despite the availability of these commercial products, they all suffer from the same problems of extremely high cost, sub-normal skin microstructure and inconsistent engraftment, especially in full thickness burns. Clinical practice for severe burn treatment has since evolved to incorporate these tissue-engineered skin substitutes, usually as an adjunct to speed up epithelization for wound closure and/or to improve quality of life by improving the functional and cosmetic results long-term. This review seeks to bring the reader through the beginnings of skin tissue engineering, the utilization of some of the key products developed for the treatment of severe burns and the hope of harnessing stem cells to improve on current practice.

In vitro characterization of human hair follicle dermal sheath mesenchymal stromal cells and their potential in enhancing diabetic wound healing.

BACKGROUND AIMS: Little is published on the characterization and therapeutic potential of human mesenchymal cells derived from hair follicle (HF) dermal sheath (DS). In this study, we isolated and characterized HF DS-mesenchymal stromal cells (DS-MSCs) with respect to the bone marrow mesenchymal stromal cells (BM-MSCs). We further tested if DS-MSC-conditioned medium (CM), like what was previously reported for BM-MSC CM, has superior wound-healing properties, in both in vitro and in vivo wound models compared with skin fibroblast CM. METHODS: DS-MSCs were isolated from HF and cultured in vitro to assess long-term growth potential, colony-forming efficiency (CFE), expression of CD surface markers and differentiation potential. The cytokine expression of DS-MSC CM was determined through an antibody-based protein array analysis. The wound-healing effects of the CM were tested in vitro with the use of human cell cultures and in vivo with the use of a diabetic mouse wound model. RESULTS: In vitro results revealed that DS-MSCs have high growth capacity and CFE while displaying some phenotypes similar to BM-MSCs. DS-MSCs strongly expressed many surface markers expressed in BM-MSCs and could also differentiate into osteoblasts, chondrocytes and adipocytes. DS-MSCs secreted significantly higher proportions of paracrine factors such as interleukin-6 (IL-6), IL-8 and growth-related oncogene. DS-MSC-CM demonstrated enhanced wound-healing effects on human skin keratinocytes, fibroblasts and endothelial cells in vitro, and the wound-healing time in diabetic mice was found to be shorter, compared with vehicle controls. CONCLUSIONS: Human HF DS stromal cells demonstrated MSC-like properties and might be an alternative source for therapeutic use in wound healing.

Breast cancer resistance protein identifies clonogenic keratinocytes in human interfollicular epidermis.

INTRODUCTION: There is a practical need for the identification of robust cell-surface markers that can be used to enrich for living keratinocyte progenitor cells. Breast cancer resistance protein (ABCG2), a member of the ATP binding cassette (ABC) transporter family, is known to be a marker for stem/progenitor cells in many tissues and organs. METHODS: We investigated the expression of ABCG2 protein in normal human epidermis to evaluate its potential as a cell surface marker for identifying and enriching for clonogenic epidermal keratinocytes outside the pilosebaceous tract. RESULTS: Immunofluorescence and immunoblotting studies of human skin showed that ABCG2 is expressed in a subset of basal layer cells in the epidermis. Flow cytometry analysis showed approximately 2-3% of keratinocytes in non-hair-bearing epidermis expressing ABCG2; this population also expresses p63, ß1 and α6 integrins and keratin 14, but not CD34, CD71, C-kit or involucrin. The ABCG2-positive keratinocytes showed significantly higher colony forming efficiency when co-cultured with mouse 3T3 feeder cells, and more extensive long-term proliferation capacity in vitro, than did ABCG2-negative keratinocytes. Upon clonal analysis, most of the freshly isolated ABCG2-positive keratinocytes formed holoclones and were capable of generating a stratified differentiating epidermis in organotypic culture models. CONCLUSIONS: These data indicate that in skin, expression of the ABCG2 transporter is a characteristic of interfollicular keratinocyte progentior cells and suggest that ABCG2 may be useful for enriching keratinocyte stem cells in human interfollicular epidermis.

Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

Keloid fibroblasts are more sensitive to Wnt3a treatment in terms of elevated cellular growth and fibronectin expression.

BACKGROUND: Current evidence suggests the potential role of Wnt signalling in keloids pathogenesis but such literature remains scanty. We hypothesize that Wnt signalling is upregulated in keloid fibroblasts (KFs) and this promotes cellular growth, migration and extracellular matrix (ECM) production in such fibroblasts. OBJECTIVES: To verify the downregulation of secreted frizzled-related protein 1 (SFRP1), a Wnt inhibitor and test KFs sensitivity to Wnt3a treatment compared to NFs in terms of activation of Wnt/ß-catenin, cellular growth, migration and ECM expressions. Next, to investigate if ectopic expression of SFRP1 and treatment of quercetin in KFs can reverse their phenotypes. METHODS: Quantitative Real-time PCR and western blotting were used to verify SFRP1 expression in NFs and KFs. The fibroblasts were tested with Wnt3a conditioned media and its effects were tested for (1) the cells' sensitivity to direct Wnt signalling via the activation of TCF reporter assay and protein expression of ß-catenin, (2) cellular growth, (3) cell migration and (4) expressions of ECM components. Finally KFs were stably transduced with SFRP1 and treated with 2 doses of quercetin. RESULTS: Lower levels of SFRP1 were confirmed at mRNA and protein levels in KFs which partly explained their sensitivity to Wnt3a treatment in terms of higher Wnt activation, cellular growth and fibronectin expression. Interestingly, Wnt3a did not promote higher cell migration rate and increase in collagen I expression. Ectopic expression of SFRP1 and quercetin treatment was able to mitigate Wnt3a-mediated phenotype of KFs. CONCLUSIONS: Using SFRP1 or inhibitors of Wnt signalling might be one of the therapeutic solutions to treat keloid scarring.