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Research Article

Human Plasma Versus Collagen as a Dermal Scaffold for the Generation of a Completely Autologous Bioengineered Skin

Stefano Negri1*, Sara Farinato1, Chiara Fila2, Debora Lepri2, Patrizia Mondini1

1C.Poma Hospital, Pathological Anatomy Service, Italy
2C.Poma Hospital, Immunohaematology and Trasfusion Medicine Service, Italy

*Corresponding author: Dr. Stefano Negri, M.D. Pathological Anatomy Service, C. Poma Hospital, Via Lago Paiolo, 46100 Mantova, Italy, Tel: +39 3803097729; Email: stene60@libero.it

Submitted: 09-01-2015 Accepted: 09-26-2015 Published: 10-09-2015

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Article


Abstract

The purpose of this work is to compare autologous plasma and collagen type I as scaffold for the growth and differentiation of keratinocytes and fibroblasts.

Our studies are focussed on the isolation of human basal keratinocytes and fibroblasts from autologous bioptic skin samples and on their proliferation in culture flasks. Autologous plasma and collagen type I are subsequently used as scaffold for the  formation of skin grafts whose morphofunctional and immunohistochemical characteristics are similar to those of normal skin.

In order to evaluate the results the samples of plasma were fixed in formalin, embedded in paraffin, stained with haematoxilyn- eosin and viewed through a microscope counting the number of cells in 5 fields with a 40X objective.

Furthermore, all specimens underwent immunohistochemical examination for vimentin, keratin AE1 and AE3, collagen IV, P63 and Mib 1.

The skin grafts obtained in our study consist of few layers of normally-shaped keratinocytes on a plasma or collagen I matrix with fibroblasts embedded inside.

The formation of basement membrane shows that autologous plasma and collagen I are a good scaffold for the growth, differentiation and expansion of keratinocytes and fibroblasts and that dermal and epidermal cells can interlock to reconstruct artificial grafts identical to normal skin.

Keywords: Plasma and Collagen I Scaffold; Keratinocyte; Fibroblast; Basement Membrane; Histology; Immunohistochemistry

Introduction

Tissue engineering is a multidisciplinary area of research which aims to regenerate damaged tissues and organs in the human body, starting from the assumption that almost all animal tissues in the human body can be cultivated in a laboratory [1].The general principle is to isolate stem cells from a patient who requires a transplant and then the cells are cultured to grow and differentiate on a suitable support to produce the replacement tissue. On the one hand, it is necessary to find a suitable support (matrix or scaffold) on which cells can adhere and form stratified structures. On the other hand, the conditions allowing cells to proliferate and differentiate into the various types of tissues must be understood and reproduced.

Tissue engineering has allowed the development of skin substitutes (bioengineered skin) with the purpose of fast cell growth and reproduction without any risk of infection [2].

Our studies are focussed on the isolation of human basal keratinocytes and fibroblasts from autologous biopsied skin samples and on their proliferation in culture flasks. Autologous plasma and collagen I are subsequently used as scaffold for the formation of skin grafts whose morphofunctional and immunohistochemical characteristics are similar to those of normal skin.

In the past, keratinocyte cultures were used for the treatment of ulcers and burns thanks to their high capacity for in vitro expansion.

However, in many cases, dermal loss poses a serious problem for the grafting of keratinocytes themselves. At present, the materials used as support in the treatment of ulcers and burns are manifold, such as plasma, collagen , hyaluronic acid, fibrin, PLGA, cadaver skin.

Consequently, we have devised a method for dermal regeneration using autologous fibroblasts immersed in a matrix of human plasma or collagen I so as to eliminate any problem linked to the safety of the sample and rejection from a patient [3-4-5- 6-7]. On this new dermis, keratinocytes have then been seeded and the interaction of fibroblasts and keratinocytes has led to the formation of the basement membrane.

Culturing keratinocytes and fibroblasts can then constitute a viable alternative to conventional therapies for reconstructive surgery in case of burns, diabetic or vascular ulcers (both arterial and venous) or ulcers due to high blood pressure. These pathologies affect elderly people chronically, and thus increase their socio-economic impact [1].

Materials and Methods

Normal cells can be kept in vitro in flasks or in Petri dishes for a duration that depends on the cell type and the conditions of the culture.

Important factors for cell cultures are the density of the seeding, the choice of medium, scaffold cells and incubation conditions. The seeding density affects cellular growth to a considerable extent as cells grow better above a certain density. This is due to interaction among cultured cells and it is likely to be connected to the production of factors such as cytokines which affect cellular proliferation.

Common mediums such as MEM (Minimal Essential Medium) and DMEM (Dulbecco Modified Eagle’s Medium) contain, besides buffers and PH indicators, inorganic salts, vitamins, nitrogenous bases, carbohydrates, lipids and aminoacids, both essential and non. These mediums need to be supplemented with varying quantities of animal serum (for example fetal bovine serum). The medium must be changed every 2 to 5 days depending on the metabolic consumption and cellular density.

The culture is often supplemented with another cellular type acting as support to the cells to be cultured so as to allow a considerably lower seeding density. In the case of keratinocytes, this function is performed by 3T3 fibroblasts (a stabilized line derived from Swiss strain embryonic mouse fibroblasts). The cultures are stored in CO2 5% incubators at 37°C. Culture sterility is ensured by the medium, sterile materials and handling in a controlled environment (vertical laminar flow cabinet) [8].

The human keratinocytes and fibroblasts used for this research were taken from a skin fragment after mastectomy in a 45-year-old patient.

The experiment was approved by the independent ethics committee of C. Poma Hospital and the patient was requested to give informed consent.

Extraction of human keratinocytes and fibroblasts

This technique entails removal of the subskin, fragmentation of the graft, immersion in alcohol at 70% for five seconds and later in PBS.

The grafts are then immersed in DMEM 1X supplemented with penicillin-streptomycin (1%) and placed in a refrigerator at 4°C for at least 2 hours. This procedure lowers the bacterial potential.

After 2 hours the tissue is transferred in Dispase all night at 4°C or for an hour at 37°C. This neutral protase acts as a type 4 collagenase and separates the dermis from the epidermis in the region of the lamina lucida. At this point, the dermis and the epidermis become separated.

Fibroblasts extraction from the dermis.

Once separated from the epidermis, the dermis must be further fragmented, immersed in collagenase (crude type I, Sigma- Aldrich) and stored in a CO2 5% incubator for 7 hours. A specific medium, supplemented with serum 25%, is prepared in order to stop the action of collagenase.

Later, the solution is filtered through a 70μm sterile filter and the cells are centrifuged at 1200 rpm for 10 minutes.

Seeding procedure of extracted fibroblasts

The cells are re-suspended in 2 ml of medium, counted in the Burker chamber, and then seeded in a flask with DMEM medium supplemented with fetal bovine serum 10%, 4mM glutamine, 50IU/ml penicillin-streptomycin and 50 ml/100 ml fungizone. The culture is stored in a CO2 5% incubator and the medium is changed every 72 hours.

Trypsinization of human fibroblasts

Once the fibroblasts have come to a confluence, the medium that contained them is removed and the flask is washed with PBS.

Then 10 ml trypsin 0.05%/EDTA 0,02% are added and the solution is placed in a thermostated bath for 5 minutes. When the fibroblasts have become detached from the flask, an equal quantity of fibroblast medium is added to block the action of trypsin.

After centrifugation at 1200rpm for 10 minutes, the pellet obtained is suspended in a suitable quantity of fibroblast medium. Finally, the cells are counted using a microscope with the Burker chamber and seeded in new flasks.

Extraction of keratinocytes from the epidermis

The epidermis is treated with trypsin 0.05%/EDTA 0.02% in a thermostated bath for 30 minutes.

A pipette should be used for 5 minutes to separate the keratinocytes. The action of trypsin is blocked with medium supplemented with serum 25%. Later, the solution is filtered with a 100 μm filter and the cells are centrifuged at 1200 rpm for 10 minutes.

Seeding procedure of extracted keratinocytes

The pellet obtained is re-suspended in a suitable quantity of keratinocyte medium. The cells are counted with the Burker chamber and then seeded in a flask with a suitable amount of Swiss strain 3T3 mouse fibroblasts irradiated with 7000 RAD (seeded the day before or on the same day as the keratinocytes). The medium employed consists of a mixture of DMEM/HAM F-12 supplemented with 4 mM glutamine, 50 IU/ml penicillin- streptomycin, insulin 6 mg/ml, transferrin 5 mg/ml, adenin 0.18mM, hydrocortisone 0.4mg/ml, triiodo-thyronine 20pM, choleric toxin 0,1nM, Eμm GF (Epidermal Growth Factor) 10ng/ml, 10% fetal bovine serum, 0.25 μg/ml of fungizone. It should be remembered that the EGF must be added only 48 hours after the beginning of the culturing process in order to synchronize cellular growth [9].

The culture is stored in a CO2 5% incubator and the medium is changed every 48 hours.

Trypsinization of human keratinocytes

Once the keratinocytes have reached sub-confluence, the medium is removed and the flask is washed with PBS.

3T3 fibroblasts are eliminated by incubation for 5 minutes with 10 ml PBS/EDTA 0.02%. After removal, and another washing with PBS, 10 ml trypsin 0.05%/EDTA 0,02% are added. After   ten/fifteen minutes storage in a CO2 5% incubator, the action of trypsin is blocked by adding an equal quantity of medium containing serum 25%.

After centrifugation, the pellet obtained is re-suspended in a suitable quantity of medium for cellular count.

The cells obtained are re-seeded in new flasks with an adequate amount of 3T3 fibroblasts.


Cryopreservation of keratinocytes and fibroblasts

If the fibroblasts and keratinocytes are not used immediately, they can be cryopreserved.

2∙106 cells are re-suspended in 900 μl medium with 100 μl of DMSO. The cells are then stored in a freezer at a temperature of -80 degrees centigrade for up to a week and subsequently immersed in liquid nitrogen.

Preparation of the Human plasma scaffold

When a suitable amount of fibroblasts and keratinocytes has been obtained, the preparation of the autologous human plasma scaffold can start.

For this purpose, 5 mg of tranexamic acid is dissolved in 5 ml of plasma and then 6.5 ml of physiological solution and 5 ml of calcium chloride (1%) are added.

At the same time the plasma matrix is supplemented with 15.104 / cm2 of fibroblasts. The suspension obtained is placed on multiwell plates which are stored in a CO2 5% incubator at 37°C for 30 minutes.

This procedure enables the plasma scaffold to solidify thanks to the presence of tranexamic acid and calcium chloride (its substrate). The combination of these 2 substances enables the  solution to acquire gel-like density.

15.104 / cm2 keratinocytes are seeded simultaneously with fibroblasts or the day after on the matrix; the scaffold is covered with a layer of culture medium and then stored in an incubator for 10-12 days to allow cellular growth. The medium is changed every 48 hours.

Results showed that cells reached their optimal growth after 13 days.

Preparation of the collagen I scaffold

The keratinocytes and fibroblasts are seeded on a 0.15 cm Type I, commercial collagen sponge (ANTEMA® SOFT, Opocrin S.p.A, Modena, Italy).

ANTEMA® SOFT, a lyophilized 1.5 mm thickness sponge, is made of pure type I collagen, extracted from equine tendons and purified according to a manufacturing method which removes immunogenic structures (telopeptides) from the protein. The scaffold is first washed with saline solution and then with PBS to obtain pH 7.

The fibroblasts are then seeded at a density of 1⋅105/cm2; contemporarily, also the keratinocytes are seeded at a density of 1.8⋅105/cm².

About 3 hours after seeding, the membrane is completely immersed by adding enough medium. The culture medium is changed every 2 days.

Histological and immunohistochemical examination

After 13 days the support with fibroblasts and keratinocytes was taken in sterile conditions and immersed in fixative liquid (10% formalin) for a minimum of two hours. The sample was then subjected to dehydration, clarification and inclusion in paraffin. 3-micron microtome sections of the sample were spread out in a thermostated water bath (37°C) and then collected in histology slides. The slides were dried, the paraffin was removed first by treatment with xylene and then with a decreasing series of alcohol concentrations (100%, 95%, 50%). Sections were then re-hydrated by passing through water.

One section was stained with haematoxylin-eosin. The section was immersed in haematoxylin for 16 minutes, washed in tap water, immersed in 95% alcohol, stained with alcoholic eosin, dehydrated in alcohol, clarified in xylene, and mounted with balsam.

Additional sections underwent immunohistochemical examination for keratin-AE1, and AE3, collagen IV, P63 and Mib1 (all antibodies of Bio-optica).

Bound primary antibodies were revealed with biotin-labeled secondary antibodies and avidin-conjugated peroxidase. Stained sections were counter-stained with haematoxylin.

Results

After 13 days of culture the fragments of plasma and collagen I underwent histochemical and immunohistochemical examination.

As we can see in Figure 1, the thickness of the two fragments is superimposed and ranges between 1 and 1.5 mm. Epidermis can be noticed on the surface while the fibroblasts settle at the bottom, immersed among the collagen fibres or in the plasma. However, as shown in Figure 2, the keratinocytes cluster only on the surface, while, if we use collagen, they sink, to the bottom, in the shape of buds.

regen fig 6.1

Figure 1. Haematossilin Eosin. Magnification x 200.

regen fig 6.2

Figure 2. Immunohistochemistry – Keratin AE3. Magnification x 200.

Immunohistochemical examination for collagen IV revealed the presence of basement membrane (Figure 3). Basal stem cells were highlighted on immunohistochemical examination for P63 (Figure 4); immunohistochemical examination for Mib 1 (Figure 5) assessed the cell proliferation index, shows how many basal cells are in mitosis.

Figure 3. Immunohistochemistry – Collagen IV. Magnification x 400 – x 600.

regen fig 6.3

Figure 4. Immunohistochemistry – P 63. Magnification x 400.

regen fig 6.4

Owing to the characteristics of the collagen membrane, it is possible to cultivate keratinocytes and fibroblasts in complete immersion so as to obtain a high replication of undifferentiated adult stem cells (Figure 6).

Melanocytes grow among the cells of basement layer with collagen I, which was never observed when plasma was used as scaffold (Figure 7).

Figure 5. Immunohistochemistry – MIB 1. Magnification x 400.

regen fig 6.5

Figure 6. Immunohistochemistry – P 63. Magnification x 800 – Collagen scaffold.

regen fig 6.6

Figure 7. Immunohistochemistry – HMB45. Magnification x 600 – Collagen scaffold.

regen fig 6.7


Discussion

Tissue bioengineering is an important innovative field of research whose purpose is the regeneration of damaged organs and tissues.

Bioengineered skin can be employed in the treatment of surgery- related skin loss such as injuries due to the removal of skin lesions (giant nevi) [10], reconstruction of critical facial areas (maxillo-facial or ear surgery) or coverage after laser-resurfacting or dermabrasion.

Moreover, bioenginnering skin can reveal itself extremely useful in case of post traumatic skin loss caused by extensive, partial or full-thickness burns, limited or full-thickness burns in critical sites (face, palmar or plantar areas) or full-thickness injuries (scalping, lacerations and contusions).

Finally, skin substitutes can be custom designed to treat skin loss due to pathologies such as Lyell syndrome, congenital epidermolysis bullosa and chronic ulcers [1,11,12].

Recently bioengineered epidermis has been successfully employed in the treatment of vitiligo [13].as the patient’s keratinocytes
and melanocytes can be cultured and expanded and then grafted in order to obtain the re-pigmentation of achromatic areas.

Furthermore, bioengineering skin can offer new opportunities for the treatment of photoaging thanks to the steady improvements in research on chronic alterations due to sun-related damage and on tissue repair techniques [14].

This therapeutical approach gives excellent results as it eliminates cells which suffered prolonged damage (exposure to light and ageing) and replaces them with undamaged cells.

The skin grafts obtained in our study consist of few layers of normally-shaped keratinocytes on a plasma or collagen I matrix with fibroblasts embedded inside.

Keratinocytes and fibroblast interact each other to maintain skin integrity. Fibroblast influence keratinocyte proliferation, differentiation and migration processes involved in epidermal homeostasis and reepithelization after epidermal damage. Fibroblast- keratinocyte interactions are involved in the formation of the basement membrane zone, which is essential for attachment of the epidermis to the dermis. The formation of basement membrane shows that autologous plasma and collagen type I are a good scaffold for the growth, differentiation and expansion of keratinocytes and fibroblasts and that dermal and epidermal cells can interlock to reconstruct artificial grafts identical to normal skin [12, 15].

In particular, we have observed that fibroblast-enriched plasma or collagen can replace dermis temporarily, besides being a robust scaffold for the growth of keratinocytes [12, 16].

The benefits of living keratinocytes in the epidermal compartment and living fibroblasts in the dermal compartment above an acellular dressing have now been widely accepted: living skin substitutes secrete a cocktail of cytokines, chemokines and growth factors that promote wound healing (by stimulating angiogenesis, granulation tissue formation and epithelialisation) as well as providing an immediate cover for the wound [17-18-19].

Recent data have also shown that the presence of autologous fibroblasts can ensure good aesthetic and functional results [20, 21].

This kind of therapy is ideal for patients with chronic defects requiring continuous grafts.

Both plasma and collagen are cheap and easy to prepare.

Plasma and collagen have all the necessary requirements to be excellent support bio materials, that is to say, materials designed to interlock with biological systems in order to treat, increase or replace any kind of tissue or organ. Both have proved
themselves to be excellent scaffolds for the proliferation and differentiation of keratinocytes and fibroblasts as they can be easily tolerated by patients and are replaced by human tissue after integration. Finally, inside plasma and collagen, cells can communicate and exchange signals both between themselves and with host cells [4,7].

The main differences between the two types of scaffold are the following:

Autologous plasma eliminates all problems connected with the rejection and/or safety of the sample. Compared to collagen, it also allows a good cellular growth with a lower seeding density (15.104 / cm2 fibroblasts and 15.104 / cm2 keratinocytes , versus 1⋅105/cm2 fibroblasts and 1.8⋅105/cm² keratinocytes).

On the other hand collagen enables cellular growth, both differentiated (in a spinosum, granulosum, corneum stratum) and undifferentiated with the formation of a larger quantity of adult stem cells. This depends on whether the collagen is completely immersed in the culture medium or the keratinocytes are left to proliferate in the liquid-air interface.

When plasma is used, melanocytes are missing, while they grow among the epidermal cells of the basal layer when the collagen membrane is employed.

We believe that the reason for this phenomenon is the greater permeability of the collagen scaffold which allows keratinocytes to sink towards the neo-derma in the shape of tokens or buds almost as it happens in “ in vivo “ cutis.

Another advantage of the collagen membrane is that it makes it unnecessary to take blood samples from a patient with burns,
which is essential for the formation of the of the plasma scaffold.

Conclusion

The purpose of this work is to compare autologous plasma and collagen type I as scaffold for the growth and differentiation of keratinocytes and fibroblasts.

Our studies are focussed on the isolation of human basal keratinocytes and fibroblasts from autologous bioptic skin samples and on their proliferation in culture flasks. Autologous plasma and collagen type I are subsequently used as scaffold for the formation of skin grafts whose morphofunctional and immunohistochemical characteristics are similar to those of normal skin.

The skin grafts obtained in our study consist of few layers of normally-shaped keratinocytes on a plasma or collagen I matrix with fibroblasts embedded inside.

Keratinocytes and fibroblast interact each other to maintain skin integrity. Fibroblast influence keratinocyte proliferation, differentiation and migration processes involved in epidermal homeostasis and reepithelization after epidermal damage. Fibroblast- keratinocyte interactions are involved in the formation of the basement membrane zone, which is essential for attachment of the epidermis to the dermis. The formation of basement membrane shows that autologous plasma and collagen type I are a good scaffold for the growth, differentiation and expansion of keratinocytes and fibroblasts and that dermal and epidermal cells can interlock to reconstruct artificial grafts identical to normal skin.

References

References

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16.Sriram G, Bigliardi PL, Bigliardi-Qi M. Fibroblast heterogeneity and its implications for engineering organotypic ski models in vitro. Eur J Cell Biol 2015, SO171- 9335(15)30006-6.

17.Hartmann- Fritsch F, Biedermann T, Braziulis E, Meuli M, Reichmann E. A new model for preclinical testing of dermal substitutes for human skin reconstruction. Peditr Surg Int. 2013, 29(5): 479-488.

18.Zoller N, Valesky E, Butting M, Hofmann M, Kipppenberger S et al. Clinical application of a tissue-cultured skin autograft: an alternative for the treatment of non-healing or slowly healing wounds? Dermatology. 2014, 229(3): 190- 198.

19.Rahmanian-Schwarz A, Held M, Knoeller T, Stachon A, Schmidt T et al. In vivo biocompatibility and biodegradation of a novel thin and mechanically stable collagen scaffold”. J Biomed Mater Res A. 2014, 102(4): 1173-1179.

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Cite this article: Negri S. Human Plasma Versus Collagen as a Dermal Scaffold for the Generation of a Completely Autologous Bioengineered Skin. J J Regener Med. 2015, 1(1): 006.

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