Research ArticleClinical Studies

Clinical Application of Autologous Three-cellular Cultured Skin Substitutes Based on Esterified Hyaluronic Acid Scaffold: Our Experience

NICOLÒ SCUDERI, TOMMASO ANNIBOLETTI, BRUNO CARLESIMO and MARIA GIUSEPPINA ONESTI
In Vivo November 2009, 23 (6) 991-1003;

Abstract

Background: The aim of this work was to present our experience in the use of autologous three-cellular cultured skin substitutes (CSS). This method represents a surgical alternative in the treatment of a variety of pathologies, including burns, ulcers, giant nevi and tumors. Patients and Methods: CSS were obtained from full-thickness skin biopsies collected after enrolment of 11 patients in a clinical trial protocol approved by the local Institutional Review Boards of the ‘La Sapienza’ University of Rome and registered in clinicaltrials.gov (ID: NCT00718978). CSS consisted of a structure made by a pluristratified epithelial cell surface with melanocytes (ratio 1/20) and a basement of fibroblasts kept together by an esterified hyaluronic acid scaffold that can be surgically manipulated and is gradually reabsorbed after implantation and substituted by the host connectival stroma. Results: At the time of withdrawal of medication, the graft take was comparable to that of autografts, whereas in the follow-up visits, loss of the epithelial layer varied markedly (from 5 to 70%) while fibroblast cellular component growth prevailed. In one patient, there was complete dermal-epidermal construct survival. Conclusion: Given the anatomical complexity of the skin, we still have a long way to go before we are able to recreate all the cellular and structural characteristics of this organ. Results are, however, gradually improving, as is demonstrated by a successful graft, which was histologically shown to have a three-dimensional structure that maintained its conformation in vivo (epithelium, basement membrane, dermis, subcutis) in one patient. The take of melanocytes improved the final esthetic outcome.

Permanent wound closure remains a limiting factor in the closure of extensive, full-thickness loss of substances. Cultured skin substitutes (CSS) have been increasingly used in plastic surgery, from the earliest attempts in 1981 by O'Connor et al. (1) up to the more recent works by Boyce et al. of 2006 (2) and Nie et al. of 2007 (3), to replace either the epidermal or dermal components of skin in separate surgical procedures, or to replace both components in a single procedure, yielding results similar to those of split-thickness skin autographs (AG). Boyce et al. (2, 4-12) have used CSS prepared from epidermal keratinocytes and dermal fibroblasts attached to collagen-glycosaminoglycan substrates to treat extensive, deep burns. Research is currently focused on two issues: overcoming the anatomical limitations of in vivo applications of CSS and looking for the most suitable biomaterial to use as a scaffold. The remaining anatomical limitations of CSS that emerge from these studies, if compared with AG, include hypopigmentation and the absence of blood vessels, glands or follicles. However, the addition of cultured epidermal melanocytes in preclinical models restored skin color, and cultured microvascular endothelial cells formed vascular analogs after grafting (13, 14).

Recent work by Liu et al. (15) showed successful grafting of pigmented skin equivalents in animal models, providing the basis for application in humans. Scuderi et al. (16) have reported the clinical use of a human skin equivalent obtained by supporing autologous keratinocytes, melanocytes and fibroblasts in a three-dimensional scaffold. The scaffold is made of a hyaluronic acid derivative that facilitates the deposition of an extracellular matrix of autologous origin. The resulting three-cellular CSS is called ‘one-step skin’ owing to the possibility it offers of using keratinocytes, melanocytes and fibroblasts in one procedure. As regards the most suitable biomaterial, the development of human skin equivalents on a hyaluronic acid scaffold is particularly noteworthy, since previous keratinocyte cultures containing collagen hydrogels proved to have some drawbacks, such as limited survival, poor resistance to contraction, faulty anchorage of the epidermis to the collagen matrix and lack of dermal support (17, 18). The first attempt to reconstruct, in vitro, autologous bioengineered skin based on a scaffold of esterified hyaluronic acid was described in 1998 (19). Currently, the most suitable devices for cell culture are those made with the esterified derivatives of hyaluronan, commercially available as HYAFF®11, HYAFF®11p100 for the biomaterial derived from the total esterification of hyaluronan and HYAFF®11p80 for the biomaterial derived from the partial (I.E. 80%) esterification of hyaluronan, in the three-dimensional configuration of non-woven mesh (NWM) (20-23). This biomaterial is completely biodegradable, immunologically inert and does not elicit complement activation (24, 25). HYAFF®11 is devoid of many of the drawbacks previously encountered with collagen scaffolds: it possesses good cell adhesiveness even in the absence of any coating, it ensures a three-dimensional structure that provides mechanical stability and easy handling, and can thus be used for a wider range of clinical applications, and it is more resistant to contraction than collagen-based materials.19 Moreover, three-dimensional scaffolds made of HYAFF®11 have been shown to support growth and differentiation of fibroblasts, keratinocytes and chondrocytes (20). The aim of this paper is to describe our experience in the use of autologous three-cellular CSS and the long-term follow-up (at least three years) of all the cases. For the purposes of this study, we seeded a mixed suspension composed of keratinocytes and melanocytes, at a ratio of 1:20, on a scaffold of esterified hyaluronic acid containing autologous fibroblasts to create a skin substitute with melanocytes. The study is based on 11 patients who sustained extensive substance losses ranging from a minimum of 64 cm2 (1 sheet applied) to a maximum of approximately 400 cm2 (6 sheets applied) (Figure 1).

Patients and Methods

Presentation of cases. Between January 2004 and June 2005, a total of 11 patients (8 women and 3 men; mean age 24.6 years, range 2-57 years) were treated in our department with autologous three-cellular CSS consisting of autologous fibroblasts, keratinocytes and melanocytes. All the patients were enrolled after having been informed of the treatment being proposed and the alternative procedures (such as skin grafts and expander flaps). All the patients were studied and treated according to a study protocol approved by the local Institutional Review Boards of the ‘La Sapienza’ University of Rome. The group of patients enrolled comprised: 5 cases of giant congenital nevi (thorax in 3 cases, left side of the trunk in 1 case and right leg in 1 case); 2 cases of tumors (left gluteal lymphangioma in 1 case and right leg giant angiolipoma in 1 case); 3 cases of cicatritial outcome (upper limb in 1 case, right leg and back in 1 case, and right lower limb in 1 case); 1 case of a trauma involving the muscular fascia of the thigh. The eligibility criteria are shown in Table I. Following the hematological tests, full thickness skin biopsies were carried out on all the patients to obtain an ellipse of full thickness skin (around 2.5 cm2). The biopsy was executed, under aseptic conditions on the skin, which was, at least macroscopically, free from lesions. The fragment of skin obtained was immediately sent to the laboratory. Cells isolation and culture, preparation of skin substitute construct, assessment of keratinocytes, melanocytes and fibroblasts cultures and autologous organoid skin preparation were carried out as described by Scuderi et al. (16).

Figure 1.
Figure 1.

A sheet (8×8 cm) of bioengineered skin (HYAFF®11p100) ready to graft.

Cell isolation and culture. The skin biopsy was transferred into a Petri-dish and disinfected by submersion in 75% ethanol for 30 s. Subcutaneous fat and deep dermis were excised from the biopsy sample, and the remaining tissue was cut into small pieces. All fragments were transferred into a centrifuge tube containing 0.25% trypsin (GIBCO BRL, Grand Island, NY, USA) and incubated at 37°C for 10 min in 5% CO2. Epidermal fragments were gently pipetted until disintegration into a single cell suspension. Cells were then counted, seeded into two 35 mm dishes (106 cells/dish) and cultured in keratinocyte serum-free medium (K-SFM; Gibco BRL). Melanocyte cultures were also assessed and cultured in melanocyte serum-free medium (Gibco BRL) containing 12-0-tetradecanoylphorbol acetate phorbol ester (TPA, 10 ng/ml) and 5% fetal bovine serum (FBS). Any melanocytes present among the primary keratinocyte cultures were subsequently isolated by gentle tripsinization, and the purity of the cultures was further assessed by immunofluorescence using specific keratinocyte and melanocyte markers. The dermal fragments obtained from the same biopsy were incubated at 37°C for 1.5 h in a pre-heated sterile collagenase solution (625 U/ml; Sigma Aldrich, Saint Louis, MO, USA). Subsequently, the fragments were gently pipetted until disintegration into a single cell suspension. Cells were counted, seeded at 105 cells/cm2 into 75 cm2 flasks and maintained in DMEM (Gibco BRL) containing 10% FBS.

Biomaterial. The biomaterials used in the present study were derived from the total esterification of hyaluronan with benzyl ester (HYAFF®11p100) and from the partial esterification of hyaluronan with benzyl ester (HYAFF®11p80, 80% esterification). The device was obtained from Fidia Advanced Biopolymer S.r.l. (Abano Terme, Italy). The biomaterial scaffold was configured as a non-woven mesh (NWM) of 20 mm-thick. In aqueous solution, it hydrates with a weight increase of about 40%, swelling to about double the original diameter. Degradation occurs by spontaneous hydrolysis of the ester bonds.

View this table:
Table I.

Study participation criteria and scaffolding used.

Preparation of skin substitute construct. Primary co-cultures of keratinocytes and melanocytes were established by seeding isolated keratinocytes and melanocytes at a ratio of 20:1. Co-cultures were then maintained in K-SFM and analyzed by immunofluorescence with DAPI and anti-tyrosinase, which selectively stains melanocytes, to verify the percentage of melanocytes present in the co-cultures. The first step in the skin substitute preparation was to resuspend the primary human fibroblasts obtained from the same biopsy in fibrin gel (Tissucol Baxter) (26) and to seed them at 2×105/cm2 into an NWM of HYAFF®11, a benzylic ester of hyaluronic acid (20, 21). Fibroblasts were left in place and cultured for about a week on the 10×10 cm pieces of HYAFF®11 NWM, in 4 ml of DMEM supplemented with 1% L-glutamine, 1% penicillin/streptomycin and sodium ascorbate (50 mg/ml), and enriched with 10% of the patient's own blood serum, until the cultures reached subconfluence. The medium was replaced every other day. Subsequently, a suspension of keratinocytes/melanocytes (2×105 cell/cm2) was seeded on the surface of the biomaterial. The medium was removed and changed into chemically defined medium K-SFM (Gibco BRL). The constructs were kept submerged in the culture medium for approximately five days. The cultures were then raised to the air-liquid interface for a further four days in order to promote the in vitro development of a keratinized epidermal surface with a stratum corneum analog (9).

Assessment of keratinocyte, melanocyte and fibroblas cultures. We first established primary cultures of keratinocytes, melanocytes and fibroblasts from the same full-thickness skin biopsy. The characteristics of the keratinocyte, melanocyte and fibroblast cultures obtained were analyzed by inverted phase-contrast microscopy. Keratinocytes were polygonal-shaped and possessed a larger nuclear-cytoplasmic ratio; melanocytes displayed a dendritic morphology or slender spindle shape, whereas fibroblasts exhibited a characteristic elongated shape and were clumped together. To further assess the purity of the cell cultures and to avoid cross-contamination from different cell types, immunofluorescence analysis for the expression of specific markers for keratinocytes or melanocytes was performed. Expression of cytokeratins was used as a marker for keratinocytes. Since melanocytes synthesize and package melanin into melanosomes, anti-tyrosinase antibody, one of the melanogenic enzymes belonging to the tyrosinase gene family of proteins, was used as a marker for melanocytes. In our keratinocyte cultures, all the cells were positive for cytokeratin expression, whereas in the melanocyte cultures all the cells were positive for tyrosinase expression. We then proceeded to establish a co-culture of keratinocytes and melanocytes on collagen-coated culture flasks, using a seeding ratio of 20:1, on the basis of the consensus average ratio. Co-cultures were then analyzed by immunofluorescence with anti-tyrosinase antibody to verify the percentage of melanocytes present in the co-cultures.

Autologous organoid skin preparation. We then established the organoid skin by reconstructing an organotypic culture with a three-dimensional human dermal-epidermal architecture using fibroblast cultures and a keratinocyte-melanocyte co-culture obtained from the patient's skin biopsies. Generally, keratinocyte and fibroblast expansion on a plastic support occurred within two weeks. In our study, in order to produce a skin substitute containing an upper layer of well-differentiated stratified keratinocytes lining a dermal-like structure of fibroblasts, cells were propagated for subsequent passages and then seeded in vitro within a biodegradable fabric made of HYAFF®11p100 and HYAFF®11p80 that served as a scaffolding support for both fibroblast and keratinocyte-melanocyte cultures and made handling in a clinical setting easier (7). The configuration of the construct was engineered to maintain polarity and to permit a feasible and reproducible production of the organoid skin. The sheets produced measured 8×8 cm and were transported in a package under controlled atmospheric conditions, with a thermal seal, positioned on a nutrient enriched agar gel. The maximum maintenance time inside the package before the grafting was approximately 72 hours. After around 30 days, the three-dimensional constructs were brought to the operating room under aseptic conditions.

View this table:
Table II.

Outcome of CSS graft in patient.

Clinical cases. Once the sheets of CSS arrived from the laboratory, the patients were admitted to our department and operated upon the following day. Eight patients were treated under general anesthesia, one patient was treated under local anesthesia, 1 one patient was treated under local anesthesia with sedation, and one patient was treated under spinal anesthesia. An accurate intra-operative hemostasis was performed in all cases. The grafted area in eight cases was the deep fascial muscular plane, in one case it was the subcutaneous adipose plane and in two cases it was the granulation tissue. Approximately 256 cm2 (mean) of bioengineered tissue were grafted (range 64 cm2-384 cm2). In the first two patients treated (Group A), sheets based on the HYAFF®11p100 scaffold were grafted (the one with the highest degree of esterification). In the patient with a giant angiolipoma of the leg, three sheets based on the HYAFF®11p100 scaffold were grafted to the lower area of the loss from the removal of the tumour, and three sheets based on the HYAFF®11p80 scaffold to the upper area of the same (Patient A-B). In the 8 remaining subsequently treated patients, we used sheets based on the HYAFF®11p80 scaffold (Group B). The total area grafted was 3,008 cm2: 832 cm2 with sheets based on the HYAFF®11p100 scaffold and 2,176 cm2 with sheets based on the HYAFF®11p80 scaffold. All the patients were treated using gauzes with paraffin and compressive moulage. Post-operative antibiotic therapy was prescribed in all cases and continued for one week postoperatively, while antithrombotic prophylaxis by subcutaneous heparin injection was also prescribed in three of the cases. The moulage was withdrawn on postoperative day (POD) 4 in one case because of clinical signs of local infection, on POD 5 in two cases, on POD 6 in two cases, on POD 7 in one case, on POD 8 in three cases, on POD 9 in one case and on POD 10 in one case (mean moulage withdrawn time POD 8). In all cases, the grafted areas were medicated on a regular by using disinfection with a solution of Dakin and physiological saline. In the two patients in Group A and Patient A-B, at every medication made after the moulage withdrawn, the grafted areas were closed using gauzes with paraffin and a bandage (‘closed therapy’). In the other eight patients (i.e. Group B), at every medication made after the moulage withdrawn, the grafted areas were left exposed to the air for some hours before closing (‘open therapy’). In 5 cases, hyaluronic acid (in spray form) was used as a hydrating agent (Connettivina® spray, Fidia Farmaceutici S.p.A. Abano Terme, Italy). In one case, local oxygen therapy was used to accelerate the healing process. Biopsies were taken from the epithelized areas in the 9 patients (Patient A-B and Group B) in whom such areas persisted and were submitted to histological examination (mean: POD 25, range: POD 19-34). All the patients, except the one with a complete epithelization, then underwent a second procedure to accelerate the surgical healing process (mean: POD 15), over an area ranging from 30% to 95% of the grafted area. Seven patients were grafted using partial thickness meshed skin grafts (Group A + Patient A-B + 4 patients in Group B), two patients in Group B underwent the application of a second layer of cultivates keratinocytes, while one patient in Group B underwent both procedures.

Results

When the medication was removed, the constructs in all eleven patients looked dry, with no essudation, and with a white, homogeneous colour indicating that the dermal and epidermal components were alive and well, even if they had not yet revascularized. Subsequently, the first two patients treated (Group A) by dermal-epidermal scaffold-based substitutes with a higher degree of esterification (HYAFF®11p100) gradually suffered an almost complete epithelial loss (keratinocytes and melanocytes) (Figure 2). We thus proceeded to use autologous cutaneous partial thickness meshed grafts over the area of loss, which were grafted on POD 25 in the first and on POD 29 in the second of these patients. Both patients were discharged from hospital 22 days after this second procedure, with skin coverage of 90%. In the third patient (Patient A-B) who had been treated with 3 HYAFF®11p100 scaffolds and 3 HYAFF®11p80 scaffolds for the same area of loss of substance (right leg giant angiolipoma), the same epithelial loss was observed as that observed in the Group A patients in the lower area of the area of loss (i.e. the area grafted using a scaffold of HYAFF®11p100), while we observed a persistence and stabilization of the keratinocytes throughout the follow-up in the upper area of the loss of substance (i.e. the area grafted using a scaffold of HYAFF®11p80), which macroscopically resulted in two completely epithelized areas (Figure 3). We therefore proceeded to use autologous cutaneous partial thickness meshed grafts with a second procedure performed on POD 30 in the non-epithelized areas. The patient was discharged from hospital 11 days after this second procedure and subsequently showed almost complete skin coverage at the follow-up visits. Encouraged by this result, we decided to use a matrix based on the scaffold with a lower degree of esterification (HYAFF®11p80) in the subsequent eight patients (Group B). After the initial take, we observed various degrees of loss of the epidermic layer, and a stabilization of the cutaneous coverage after 2-3 weeks ranging from 45% to 95% of the grafted area. We then used a second procedure to treat the non-epithelized areas in all the patients, except the one with a complete epithelization, which consisted in the application of partial thickness meshed (1:4) skin grafts in four cases, in the application of another layer of cultivated keratinocytes in two cases and in the combination of these two procedures in one case, all of which were performed between POD 19 and 32 (mean: POD 25). One patient (with gluteal lymphangioma), in whom we observed a superinfection from Staphylococcus aureus, was treated with specific antibiotic therapy, which resolved the infection in 4 days; this patient's clinical course was subsequently comparable to that of the others.

Figure 2.
Figure 2.
Figure 2.
Figure 2.

Patient affected by a giant congenital nevus of the left side of the trunk (Group A). She received a graft with a second procedure using partial thickness meshed skin grafts at POD 29. A, B, preoperative view; C-E, intraoperative: sheets of bioengineered skin (HYAFF®11p100) during and after grafting; F, postoperative: after 7 days the sheets are still in situ and the keratinocytes are well preserved; G, H, postoperative: after 14 and 20 days, respectively, keratinocytes are almost completely absent while there is a complete take of the fibroblasts, as confirmed histologically; I, histological appearance after 20 days, there are fibroblasts and various vacuoli due to the reabsorption of the residual part of HYAFF®11p100, there are no keratinocytes, original magnifications x5; J, postoperative: after 7 days of the grafting of partial skin meshed grafts; K, postoperative: result after 4 months. L, M, postoperative: result after 3 years.

Figure 3.
Figure 3.
Figure 3.

Patient affected by giant angiolipoma of the right leg (Patient A-B). He received a graft with a second procedure using partial thickness meshed skin grafts at POD 30 in the non-epithelized areas. A, Preoperative view; B, intraoperative view; C, sheets of bioengineered skin (HYAFF®11p100 and HYAFF®11p80) during grafting; D, postoperative: after 14 days there are no keratinocytes left where sheets of HYAFF®11p100 were grafted; E, postoperative: after 14 days there are well-epithelized areas (yellow circle) where sheets of HYAFF®11p80 were grafted; F, hematoxylin and eosin staining of skin biopsy, histological appearance of the epithelized areas after 20 days, showing the epithelium with keratinocytes, a well-developed basement membrane and some residual of scaffold that had not yet been reabsorbed, original magnifications x5; G, hematoxylin and eosin staining of skin biopsy, histological appearance of the epithelized areas after 30 days, showing the scaffold to be almost completely reabsorbed and the dermal-epidermal junction with various physiological characteristics, original magnifications x5; H, grafting of partial skin meshed grafts on POD 30; I, postoperative: result after 5 months; J, postoperative; result after 1 year showing less hypertrophy and a more homogeneous skin colour.

Biopsies were taken from the clinically epithelized grafted areas in all the patients in Group B (treated using the HYAFF®11p80 matrix) and were submitted to histological examination (mean: POD 25, range POD 19-32). All the specimens displayed the formation of typical epidermal layers, including a stratum corneum with the basement membrane similar to the physiological, even though some important anatomical structures, such as follicular bulbs, sweat glands, sebaceous glands and vascular plexi, were missing. Healing in the seven patients in this group who underwent a second procedure was almost complete at the time of discharge from hospital. All the patients (11) were followed up for three years at the outpatient clinic, the first follow-up visit being performed after two months. The follow-up visits, which were subsequently performed 4 and 8 months and 1 and 3 years postoperatively, showed perfect stabilization of the new skin areas with a progressive improvement in the cicatritial outcomes, no alterations in the cellular turnover of the more superficial layers, and good esthetic and functional results. In nine patients (Patient A-B and Group B), i.e. all those treated except those in Group A, biopsies were taken from the CSS areas during the follow-up (mean: 18 months) and at histological examination showed the presence of a highly organized skin with the basement membrane similar to the physiological, as well as the presence of melanocytes inside the new tissue at the basal layer level. The patient in Group B treated for a giant congenital nevi of the thorax, in whom a complete CSS epithelization was observed, did not undergo a second procedure and had stable results throughout the follow-up, with an outcome that was esthetically and functionally comparable to those yielded by autologous cutaneous partial thickness meshed grafts. She was discharged from hospital on POD 18 and followed up for three years at the outpatient clinic, with the first visit after two months. After 18 months, the cellular organization in the skin sample displayed a regular multilayered epithelial structure with normal development and regeneration. The skin also showed organized connective tissue in the dermis and a well-defined cornified layer. Furthermore, there were no major signs of retraction or limitation in upper limb or neck mobility: the scar was not painful either spontaneously or on palpation. The grafted tissue provided good cover for the deep structures and had good mechanical properties. No corneal desquamation was observed and the new tissue was well hydrated. The total area of graft skin required in this patient (in relation to the area treated) was significantly smaller than that required for the other patients (Figure 4, Table II).

Figure 4.
Figure 4.

Patient affected by a giant congenital nevus of the thorax. She did not undergo a second procedure. A, preoperative view; B, postoperative: after 15 days the sheets are still in situ with keratinocytes that are well preserved; C, D, hematoxylin and eosin staining of skin biopsies, histological appearance after 20 and 30 days showing the evolution towards well-differentiated skin layers, original magnifications x5; E, postoperative: result after 6 months showing areas of hypertrophy of the scars, especially at the level of the junction between the sheets and in the areas subject to traction forces (interscapular area); F, hematoxylin and eosin staining of skin biopsy, histological appearance after 18 months, original magnifications x10; G, postoperative: result after 3 years showing a reduction in the hypertrophy and that the skin is normochromic.

Discussion

Some interesting structural (i.e. the degree of esterification of the sheets used) and clinical data emerge from this study, particularly as regards the procedure performed, the patients' ages, the therapy used (open or closed and the disinfectant) and the best supporting tissue for the graft. We used a procedure based on the use of a scaffold made of non-woven, fibrous, esterified hyaluronic acid that has far fewer drawbacks than CSS based on a collagen scaffold. Thanks to the support material, which is totally biocompatible and non-immunogenic, the construct has a three-dimensional structure that assures good mechanical stability, good mechanical resistance to contraction and easy handling, thus allowing clinical applications over wide areas.

Previous in vitro studies have shown hyaluronan derivatives with a lower percentage of esterification to have a faster degradation (27). In contrast to the findings reported by Price et al. in 2006 (28), we obtained better results using a matrix based on the scaffold with a lower degree of esterification (HYAFF®11p80), as opposed to that with a higher degree of esterification (HYAFF®11p100); after the first two patients, in whom we used the HYAFF®11p100 scaffold, we used the HYAFF®11p80 scaffold in all the subsequent patients, who were also treated by seeding the autologous fibroblasts with a fibrin gel. We presume that the matrix of hyaluronic acid with a lower degree of esterification (p80) degrades faster, allowing the keratinocytes to be nourished more rapidly, thus permitting their survival, particularly in the initial phases of take, and that the presence of fibrin in the matrix accelerates the formation of the connections between the graft and the host tissue. We observed that the muscular fascia used as a supporting tissue for the organotypic bioengineered skin graft is more effective than a layer of subcutaneous adipose tissue or granulation tissue, which are less vascularized. Similarly, bipolar coagulation for hemostasis is preferable to monopolar coagulation because it causes less thermal damage to the tissue that is to be grafted. As regards the therapy, we first immobilized the sheets by means of an occlusive-compressive moulage to start the process of integration between the tissue cultivated in vitro and the muscular fascia, then removed it to expose the graft to the air (open therapy), a technique that yielded better results than the closed method, probably owing to a mechanism that allows the differentiation of the keratinocytes into the protective stratum corneum (29-31). The use of mildly histolesive disinfectants (solution of Dakin) and the exposure to the air accelerated the process of stabilization of the grafts, as did the use of hyaluronic acid (in spray form) for hydrating (Connettivina® spray, Fidia Farmaceutici S.p.A. Abano Terme, Italy) and, in one patient, of local oxygen therapy.

We observed a better proliferation of keratinocytes in younger patients. Moreover, the development of autologous three-cellular CSS that harbour melanocytes besides keratinocytes and fibroblasts seems to yield better aesthetic results than skin equivalents derived from keratinocyte and fibroblast co-cultures alone, in which the pigmentation remains whitish after grafting (2,9). In this study, we selected a ratio of melanocytes to keratinocytes of 1:20 based on the ratio found in the human epidermis across all ethnicities, and we seeded a mixed suspension composed of keratinocytes and melanocytes. We believe that the use of this mixed suspension affects the skin pigmentation and allows the creation of a pigmented epidermis that yields a better outcome than cultures based on fibroblasts and keratinocytes alone.

The results achieved with the in vivo experimentation of autologous three-cellular CSS on a scaffold of HYAFF®11 confirm that the use of in vivo organotypic complex structures made in the laboratory is feasible. On the basis of these results and on the clinical experience acquired using monocellular bioengineered tissues, we started developing and using organotypic co-cultures of autologous fibroblasts, keratinocytes and melanocytes. We observed, after an initial take of the new structure, that the loss of keratinocytes varied in patients. Although the underlying mechanisms are still unknown, they appear to be related to the excessive growth of fibroblasts, which, when combined with other factors, limit the nourishment or O2 required for keratinocyte survival, which in turn highlights the importance of maintaining a well-balanced growth of these two cellular components. A better knowledge of these mechanisms may in the future improve the percentage of take of these sheets by limiting the excessive growth of the fibroblasts, thus favouring that of the keratinocytes. The first successes, with a complete sheet take being achieved in one case, point to the need for a close, constant collaboration between researchers and clinicians; moreover, both the bioengineered structure and the clinical protocols also need to be constantly improved to achieve better clinical results.

The three-cellular CSS lacks some important anatomical structures, such as follicular bulbs, sweat glands, sebaceous glands and vascular plexi. The next target in tissue bioengineering will be a cutaneous substitute in which the dermis and epidermis are combined with other cellular components, such as endothelial vessel cells, which may adequately nourish the new tissue through a capillary vessel network. Vascularization is essential for the complete take of cutaneous substitutes, as it is for all the types of grafts. Recently, the development of skin equivalents has further improved thanks to the creation of a dermal-epidermal architecture including a microcapillary network in a three-dimensional biomaterial (15, 32, 33), which addresses the issue of the vascularization of skin substitutes that is critical to all kind of grafts. Greater knowledge of the mechanisms underlying the start of angiogenesis during tissue culture in vitro will be able to reduce the vascularization times of the bioengineered graft, and will consequently improve the take percentage of these sheets. Retraction and cicatritial hypertrophy are another two issues that deserve further investigation in patients treated with organotypic bioengineered skin. Clinical findings from autologous skin grafts show that the greater the thickness of the deep dermic component, the less the retraction. Some components of the deep derma appear to be able to suppress the activity of myofibroblasts, the cells involved in retraction, through an unknown mechanism (34). The total area of donor skin required in patients with higher percentages of CSS take was lower than that required when meshed skin grafts were used on their own.

Conclusion

The clinically based results yielded by our study are encouraging; though they need to be confirmed by further studies with more standardized protocols as well as a longer follow-up. Once these results are shown to reproducible and stable, this new bioengineered skin graft approach may become an alternative to the other procedures.

Aknowledgements

We thank the Fidia Advanced Biopolymer S.r.l. and Fidia Farmaceutici S.p.A (Abano Terme, Italy) for the Biomaterial furnished to our clinical trial.

  • Received March 16, 2009.
  • Revision received August 6, 2009.
  • Accepted September 18, 2009.

References

  1. ↵
    1. O'Connor NE,
    2. Mulliken JB,
    3. Banks-Schlegel S,
    4. Kehinde O,
    5. Green H
    : Grafting of burns with cultured epithelium prepared from autologous epidermal cells. Lancet 1(8211): 75-78, 1981.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Boyce ST,
    2. Kagan RJ,
    3. Greenhalgh DG,
    4. Warner P,
    5. Yakuboff KP,
    6. Palmieri T,
    7. Warden GD
    : Cultured skin substitutes reduce requirements for harvesting of skin autograft for closure of excised, full-thickness burns. J Trauma 60(4): 821-829, 2006.
    OpenUrlPubMed
  3. ↵
    1. Nie X,
    2. Zhang JY,
    3. Cai KJ,
    4. Yang MH,
    5. Xiao AH,
    6. Da Hu H,
    7. Liu LY,
    8. Wang HJ,
    9. Wen N,
    10. Jin Y
    : Cosmetic improvement in various acute skin defects treated with tissue-engineered skin. Artif Organs 31(9): 703-710, 2007.
    OpenUrlPubMed
  4. ↵
    1. Supp DM,
    2. Karpinski AC,
    3. Boyce ST
    : Expression of human beta defensins HBD-1, HBD-2, and HBD-3 in cultured keratinocytes and skin substitutes. Burns 30(7): 643-648, 2004.
    OpenUrlPubMed
    1. Boyce ST,
    2. Warden GD
    : Principles and practices for treatment of cutaneous wounds with cultured skin substitutes. Am J Surg 183(4): 445-456, 2002.
    OpenUrlCrossRefPubMed
    1. Boyce ST,
    2. Supp AP,
    3. Harriger MD,
    4. Pickens WL,
    5. Wickett RR,
    6. Hoath SB
    : Surface electrical capacitance as a noninvasive index of epidermal barrier in cultured skin substitutes in athymic mice. J Invest Dermatol 107(1): 82-87, 1996.
    OpenUrlCrossRefPubMed
  7. ↵
    1. Boyce ST,
    2. Supp AP,
    3. Swope VB,
    4. Warden GD
    :Vitamin C regulates keratinocyte viability, epidermal barrier, and basement membrane formation in vitro, and reduces wound contraction after grafting of cultured skin substitutes. J Invest Dermatol 118(4): 565-572, 2002.
    OpenUrlCrossRefPubMed
    1. Boyce ST,
    2. Kagan RJ,
    3. Meyer NA,
    4. Yakuboff KP,
    5. Warden GD
    : Cultured skin substitutes combined with Integra to replace native skin autograft and allograft for closure of full-thickness burns. J Burn Care Rehabil 20(6): 453-461, 1999. Comment in: J Burn Care Rehabil 22(2): 197-198, 2001, J Burn Care Rehabil 22(2): 198-199, 2001.
    OpenUrlPubMed
  9. ↵
    1. Boyce ST,
    2. Kagan RJ,
    3. Yakuboff KP,
    4. Meyer NA,
    5. Rieman MT,
    6. Greenhalgh DG,
    7. Warden GD
    : Cultured skin substitutes reduce donor skin harvesting for closure of excised, full-thickness burns. Ann Surg 235(2): 269-279, 2002.
    OpenUrlCrossRefPubMed
    1. Hansbrough JF,
    2. Boyce ST,
    3. Cooper ML,
    4. Foreman TJ
    : Burn wound closure with cultured autologous keratinocytes and fibroblasts attached to a collagen-glycosaminoglycan substrate. JAMA 262(15): 2125-2130, 1989.
    OpenUrlCrossRefPubMed
  11. ↵
    1. Passaretti D,
    2. Billmire D,
    3. Kagan R,
    4. Corcoran J,
    5. Boyce S
    : Autologous cultured skin substitutes conserve donor autograft in elective treatment of congenital giant melanocytic nevus. Plast Reconstr Surg 114(6): 1523-1528, 2004.
    OpenUrlPubMed
  12. ↵
    1. Boyce ST,
    2. Glatter R,
    3. Kitzmiller WJ
    : Treatment of chronic wounds with cultured skin substitutes: a pilot study. Wounds 7: 24-29, 1995.
    OpenUrl
  13. ↵
    1. Supp DM,
    2. Supp AP,
    3. Bell S,
    4. Boyce ST
    : Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor. J Invest Dermatol 114(1): 5-13, 2000.
    OpenUrlCrossRefPubMed
  14. ↵
    1. Supp DM,
    2. Bell SM,
    3. Morgan JR,
    4. Boyce ST
    : Genetic modification of cultured skin substitutes by transduction of human keratinocytes and fibroblasts with platelet-derived growth factor-A. Wound Repair Regen 8(1): 26-35, 2000.
    OpenUrlCrossRefPubMed
  15. ↵
    1. Liu Y,
    2. Suwa F,
    3. Wang X,
    4. Takemura A,
    5. Fang YR,
    6. Li Y,
    7. Zhao Y,
    8. Jin Y
    : Reconstruction of a tissue engineered skin containing melanocytes. Cell Biol Int 31(9): 985-990, 2007.
    OpenUrlPubMed
  16. ↵
    1. Scuderi N,
    2. Onesti MG,
    3. Bistoni G,
    4. Ceccarelli S,
    5. Rotolo S,
    6. Angeloni A,
    7. Marchese C
    : The clinical application of autologous bioengineered skin based on a hyaluronic acid scaffold. Biomaterials 29(11): 1620-1629, 2008.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Stark HJ,
    2. Willhauck MJ,
    3. Mirancea N,
    4. Boehnke K,
    5. Nord I,
    6. Breitkreutz D,
    7. Pavesio A,
    8. Boukamp P,
    9. Fusenig NE
    : Authentic fibroblast matrix in dermal equivalents normalises epidermal histogenesis and dermoepidermal junction in organotypic co-culture. Eur J Cell Biol 83(11-12): 631-645, 2004.
    OpenUrlCrossRefPubMed
  18. ↵
    1. Stark HJ,
    2. Szabowski A,
    3. Fusenig NE,
    4. Maas-Szabowski N
    : Organotypic cocultures as skin equivalents: a complex and sophisticated in vitro system. Biol Proced Online 6: 55-60, 2004.
    OpenUrlCrossRefPubMed
  19. ↵
    1. Zacchi V,
    2. Soranzo C,
    3. Cortivo R,
    4. Radice M,
    5. Brun P,
    6. Abatangelo G
    : In vitro engineering of human skin-like tissue. J Biomed Mater Res 40(2): 187-194, 1998.
    OpenUrlCrossRefPubMed
  20. ↵
    1. Campoccia D,
    2. Doherty P,
    3. Radice M,
    4. Brun P,
    5. Abatangelo G,
    6. Williams DF
    : Semisynthetic resorbable materials from hyaluronan esterification. Biomaterials 19(23): 2101-2127, 1998.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Galassi G,
    2. Brun P,
    3. Radice M,
    4. Cortivo R,
    5. Zanon GF,
    6. Genovese P,
    7. Abatangelo G
    : In vitro reconstructed dermis implanted in human wounds: degradation studies of the HA-based supporting scaffold. Biomaterials 21(21): 2183-2191, 2000.
    OpenUrlCrossRefPubMed
    1. Aigner J,
    2. Tegeler J,
    3. Hutzler P,
    4. Campoccia D,
    5. Pavesio A,
    6. Hammer C,
    7. Kastenbauer E,
    8. Naumann A
    : Cartilage tissue engineering with novel nonwoven structured biomaterial based on hyaluronic acid benzyl ester. J Biomed Mater Res 42(2): 172-181, 1998.
    OpenUrlCrossRefPubMed
  23. ↵
    1. Brun P,
    2. Abatangelo G,
    3. Radice M,
    4. Zacchi V,
    5. Guidolin D,
    6. Daga Gordini D,
    7. Cortivo R
    : Chondrocyte aggregation and reorganization into three-dimensional scaffolds. J Biomed Mater Res 46(3): 337-346, 1999.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Benedetti L,
    2. Cortivo R,
    3. Berti T,
    4. Berti A,
    5. Pea F,
    6. Mazzo M,
    7. Moras M,
    8. Abatangelo G
    : Biocompatibility and biodegradation of different hyaluronan derivatives (HYAFF) implanted in rats. Biomaterials 14(15): 1154-1160, 1993.
    OpenUrlCrossRefPubMed
  25. ↵
    1. Cortivo R,
    2. Brun P,
    3. Rastrelli A,
    4. Abatangelo G
    : In vitro studies on biocompatibility of hyaluronic acid esters. Biomaterials 12(8): 727-730, 1991.
    OpenUrlCrossRefPubMed
  26. ↵
    1. Rhodes AR,
    2. Wood WC,
    3. Sober AJ,
    4. Mihm MC Jr.
    : Nonepidermal origin of malignant melanoma associated with a giant congenital nevocellular nevus. Plast Reconstr Surg 67(6): 782-790, 1981.
    OpenUrlPubMed
  27. ↵
    1. Avitabile T,
    2. Marano,
    3. Castiglione F,
    4. Bucolo C,
    5. Cro M,
    6. Ambrosio L,
    7. Ferrauto C,
    8. Reibaldi A
    : Biocompatibility and biodegradation of intravitreal hyaluronan implants in rabbits. Biomaterials 22(3): 195-200, 2001.
    OpenUrlPubMed
  28. ↵
    1. Price RD,
    2. Das-Gupta V,
    3. Leigh IM,
    4. Navsaria HA
    : A comparison of tissue-engineered hyaluronic acid dermal matrices in a human wound model. Tissue Eng 12(10): 2985-2995, 2006.
    OpenUrlCrossRefPubMed
  29. ↵
    1. Rosdy M,
    2. Clauss LC
    : Terminal epidermal differentiation of human keratinocytes grown in chemically defined medium on inert filter substrates at the air-liquid interface. J Invest Dermatol 95(4): 409-414, 1990.
    OpenUrlCrossRefPubMed
    1. Fartasch M,
    2. Ponec M
    : Improved barrier structure formation in air-exposed human keratinocyte culture systems. J Invest Dermatol 102(3): 366-374, 1994.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Bernstam LI,
    2. Vaughan FL,
    3. Bernstein IA
    : Stratified cornified primary cultures of human keratinocytes grown on microporous membranes at the air-liquid interface. J Dermatol Sci 1(3): 173-181, 1990.
    OpenUrlCrossRefPubMed
  32. ↵
    1. Supp DM,
    2. Wilson-Landy K,
    3. Boyce ST
    : Human dermal microvascular endothelial cells form vascular analogs in cultured skin substitutes after grafting to athymic mice. FASEB J 16(8): 797-804, 2002.
    OpenUrlAbstract/FREE Full Text
  33. ↵
    1. Tonello C,
    2. Vindigni V,
    3. Zavan B,
    4. Abatangelo S,
    5. Abatangelo G,
    6. Brun P,
    7. Cortivo R
    : In vitro reconstruction of an endothelialized skin substitute provided with a microcapillary network using biopolymer scaffolds. FASEB J 19(11): 1546-1548, 2005.
    OpenUrlAbstract/FREE Full Text
  34. ↵
    1. Rudolph R
    : The effect of skin graft preparation on wound contraction. Surg Gynecol Obstet 142: 49-56, 1976.
    OpenUrlPubMed
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Clinical Application of Autologous Three-cellular Cultured Skin Substitutes Based on Esterified Hyaluronic Acid Scaffold: Our Experience
NICOLÒ SCUDERI, TOMMASO ANNIBOLETTI, BRUNO CARLESIMO, MARIA GIUSEPPINA ONESTI
In Vivo Nov 2009, 23 (6) 991-1003;
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