Autologous mesenchymal stem cells culture from adipose tissue for treatment of facial rhytids

Abstracts

OBJECTIVE: To test the effect of mesenchymal stem cells (MSC) from adipose tissue on the dermal filling for nasolabial rhytids correction. MEHTODS: 50 cc of infraumbilical fat and 20 ml of peripheral blood were harvested to isolate MSC and autologous plasma from 15 female volunteers, respectively. The volunteers were grouped in according to the following strategies of intra-dermal injection: Group (I) only hyaluronic acid; Group (II) only MSC; Group (III) MSC combined with hyaluronic acid. For this qualitative and prospective study photographic monitoring was done monthly. RESULTS: In the group (I) we observed an immediate effect of filling; in the group (II) the effect of filling was reached after approximately two months. In the group (III) filling occurred more efficiently and progressively, probably due to the combination of the short and long-term effects generated by the hyaluronic acid and MSC, respectively. CONLCUSION: MSC when combined with hyaluronic acid were able to fill in deep folds, with progressive improvement of skin tone and decreasing lines of expression.

Mesenchimal stem cells; Adipose tissue; Rejuvenation

OBJETIVO: Testar o efeito das c élulas tronco mesenquimais (CTM) de tecido adiposo no preenchimento cutâneo de rítides na região naso-labial. MÉTODOS: Foram coletados 50 cc de gordura da região infra-umbilical e 20 mL de sangue periférico de 15 voluntárias do sexo feminino para obtenção das CTM e de plasma autólogo, respectivamente. As voluntárias foram agrupadas de acordo com as estratégias de injeções intra-dérmicas: grupo (1) somente o ácido hialurônico; grupo (2) somente as CTM; grupo (3) CTM associadas ao ácido hialurônico. Tratando-se de um estudo prospectivo e qualitativo o acompanhamento das voluntárias era mensal através de fotografias. RESULTADOS: No grupo (1) foi observado um efeito de preenchimento imediato ao contrário do grupo (2) onde o efeito de preenchimento pleno foi alcançado aproximadamente após dois meses. No grupo (3) o preenchimento ocorreu de maneira mais efetiva e também progressiva, devido à combinação dos efeitos de curto e de longo prazo gerados pelo ácido hialurônico e pelas CTM, respectivamente. CONCLUSÃO: As CTM quando associadas ao ácido hialurônico foram capazes de promover o preenchimento de sulcos profundos, com melhora progressiva do tônus da pele e diminuição das linhas de expressão.

Células-tronco mesenquimais; Tecido adiposo; Rejuvenescimento

ORIGINAL ARTICLE

Autologous mesenchymal stem cells culture from adipose tissue for treatment of facial rhytids

César Claudio-da-Silva, TCBC-RJ^I; Leandra Santos Baptista^II; Rosana Bizon Vieira Carias^III; Hélio da Cunha Menezes Neto^III; Radovan Borojevic^II

^IAssociate Professor, Department of General Surgery, Plastic Surgery Service, Hospital Universitário Clementino Fraga Filho, Universidade Federal do Rio de Janeiro (UFRJ), RJ, Brazil. Full Member, Sociedade Brasileira de Cirurgia Plástica - SBCP (Brazilian Society of Plastic Surgery)

^IIFellow, APABCAM, Hospital Universitário Clementino Fraga Filho, UFRJ, RJ, Brazil

^IIILaboratory Manager, Excellion Serviços Biomédicos S/A, RJ, Brazil

^IVFull Professor, Instituto de Ciências Biomédicas, UFRJ, RJ, Brazil

Correspondence address

ABSTRACT

OBJETIVE: To test the effect of mesenchymal stem cells (MSC) from adipose tissue on the dermal filling for nasolabial rhytids correction.

METHOD: 50 cc of infraumbilical fat and 20 ml of peripheral blood were harvested to isolate MSC and autologous plasma from 15 female volunteers, respectively. The volunteers were grouped in according to the following strategies of intra-dermal injection: Group (I) only hyaluronic acid; Group (II) only MSC; Group (III) MSC combined with hyaluronic acid. For this qualitative and prospective study photographic monitoring was done monthly.

RESULTS: In the group (I) we observed an immediate effect of filling; in the group (II) the effect of filling was reached after approximately two months. In the group (III) filling occurred more efficiently and progressively, probably due to the combination of the short and long-term effects generated by the hyaluronic acid and MSC, respectively.

CONCLUSION: MSC when combined with hyaluronic acid were able to fill in deep folds, with progressive improvement of skin tone and decreasing lines of expression.

Key words: Mesenchymal stem cells. Adipose tissue. Rejuvenation.

INTRODUCTION

The literature provides a variety of reports with varying degrees of success and failure of the liposculpting procedure. Success depends directly on the techniques used for harvest, cleansing and reinjection of the material¹. However, a standard protocol has not been established as yet to define the best way of processing the fat for a long-term graft regardless of the injected area².

Many physicians believe that adipocyte survival is one of the main factors having direct bearing on graft success³. However, the stromal fraction of the adipose tissue is composed of preadipocytes, endothelial cells, macrophages, fibroblasts⁴ as well as mesenchymal stem cells (MSC) that, when properly stimulated, originate bone, cartilage and adipocytes^5,6. An adult stem cell is defined by several traits, including its ability to "self-renew". When a stem cell is stimulated to divide, it can originate daughter cells that are more committed to forming a cell type from the tissue where they reside. Other daughter cells can remain at the stem cell stage and further divide countless times⁷.

Regenerative strategies based on the use of adipose tissue MSC hold great promise for medical procedures of soft tissue augmentation as they afford the possibility of long-term filling⁸. It has recently been shown that the association of MSC and fat grafting for breast augmentation promotes satisfactory clinical outcomes without further complications⁹.

In addition to the combination with tissue grafting, MSC can be associated with growth factors and/or scaffolds (synthetic or biogenic) in a process termed "tissue engineering". Hyaluronic acid is a major polysaccharide, the main component of the extracellular matrix of fetal tissues, and is present in adults as a structural component of skin and connective tissue. It also promotes cell migration and proliferation, and its rapid tissue replacement in vivo by collagen makes this polysaccharide an excellent candidate for soft tissue filling¹⁰.

The present clinical study is based on the use of tissue engineering capabilities and involves the creation of an autologous implant of MSC associated with hyaluronic acid for the dermal filling of rhytids in the nasolabial region. With that combination, the formation of a three-dimensional matrix network in vivo is expected. That network, which is hydrophilic and permeable to the nutrient-rich biological fluids, will be able to promote a favorable environment for cell growth and the production of new extracellular matrix, thereby controlling cell migration to other sites.

METHODS

The project for this prospective and qualitative study was approved on July 20, 2006 by the Research Ethics Committee of the Hospital Pró-Cardíaco (PROCEP) in the city of Rio de Janeiro under Directive 170.

In the period from August through September 2006, infraumbilical fat was harvested from 15 female volunteers, yielding 50 cc of aspirate. Additionally, 20 mL of peripheral blood were collected in order to obtain autologous plasma. Informed consent was obtained from all volunteers. The volunteers' mean age was 45.8 ± 7.48 years (mean ± standard deviation).

For the lipoaspirate harvest, asepsis and antisepsis were ensured; subsequently, 50 cc of a 0.25% lidocaine solution with 1:500,000 adrenaline were administered at the marked sites in the infraumbilical region. The material was aspirated through syringes with cannulas 3-mm in diameter, which were then sealed and placed in a decanter for 30 min for the removal of blood serum content; 10 cc of an antibiotic solution were added to the adipose tissue harvested. The material was transported in a cooled container to the processing unit.

In order to obtain MSC, the adipose tissue was processed as previously described^5,6. The cells were cultured in Dulbecco's Modified Eagles Medium (DMEM; LGC, Cotia, SP) supplemented with 10% fetal calf serum (Cultilab, Campinas, SP) and antibiotics (100 U/mL penicillin sodium and 100 mg/mL streptomycin), and kept in an oven at 37ºC with 5% CO₂ for up to one week. Two days prior to the injection session, the cells were thoroughly washed with normal saline and maintained in autologous plasma to eliminate internalized fetal calf proteins ¹¹.

On the day of the application, the cells were dissociated with 0.125% trypsin solution (Gibco BRL, Rockville, MD) and 0.78 mM EDTA (Invitrogen, Gibco) and resuspended in saline with or without hyaluronic acid (L.E.A, Paris, France) at the concentration of 1:2 for a final volume of 0.6 mL. The volunteers were randomly grouped according to the application strategies: group 1 - only hyaluronic acid, with four patients; group 2 - only adipose tissue MSC, with six patients; group 3 - adipose tissue MSC combined with hyaluronic acid, with five patients (Table 1). All injection procedures were performed on the nasogenian folds.

Since the present study was both prospective and qualitative, photographs of the patients' faces were taken at the doctor's office for up to one year on a monthly basis.

RESULTS

In group 1, an immediate filling effect was observed (Figure 1A - D). The stability of the implant varied from case to case, and total resorption occurred in volunteer 3 after 12 months (Figure 1B).

In group 2, the MSC were able to produce slight improvements in the tone of the nasolabial region (Figure 1E - H), but very deep folds were not filled out (Figure 1E, F). Overall, the effect of filling was maintained for up to 10 months (Figure 1F, H).

In group 3, there was a combination of the immediate effect of hyaluronic acid with the long-term effect of MSC. Thus, the filling of the fold was effective, and instead of the progressive resorption observed in group 1, the nasolabial region exhibited an improvement in tone over time (Figure 1I - L). No reduction in the improvement occurred in any of the three volunteers with up to one year follow-up (Figure 1J, L).

No side effects or adverse reactions were observed during the study that could be attributed to the filling agent and/or to the adipose tissue MSC.

DISCUSSION

Various types of fillers exist, natural or synthetic, each indicated for a particular type of desired repair. With respect to dermal filling, the most frequently used filling agents are collagen, hyaluronic acid, poly-L-lactic acid (PLLA), polymethylmethacrylate and some calcium hydroxyapatite gels. Because some synthetic materials are inert, they are not absorbed, hence they provide stable and lasting dermal filling. Nonetheless, some complications due to chronic inflammatory reactions have already been reported. Polymethylmethacrylate is largely responsible for those complications, which became permanent or are still unresponsive to the treatments¹².

Hyaluronic acid, used in the present study, is a natural polysaccharide found in the extracellular matrix of connective tissue, synovial fluid and other tissues. Safety is ensured by its biocompatibility, while its major drawback is resorption (6-12 months). One of the strategies to enhance its durability is the cross-linking of the molecule, which makes hydrogel more insoluble and, consequently, less resorbable¹³. Another strategy that has already been used in clinical practice is the combination of the acid with dextranomer microspheres, a glucose-derived molecule that seemingly stimulates collagen synthesis. However, a clinical case has recently been published of a diffuse granulomatous reaction at the site of the injection of the copolymer of hyaluronic acid with dextranomer for the correction of rhytids¹⁴.

The foundation for cell-based therapy protocols resides in the fact that healthy cells stimulate the tissue in which they were injected for the regeneration process¹⁵. In the filling of rhytids and acne scars, a number of protocols using skin autologous fibroblasts have already been established^16-18. In the case of cell-based therapies with stem cells, in addition to the stimulatory effect on regeneration, clinical trials report the differentiation of those cells into tissue-resident cells¹⁵. Adipose tissue is a very attractive source of MSC in cell therapy protocols in that it can be obtained in large amounts by repeat liposuction techniques⁴. In 1999, a study was conducted on the filling of rhytids using adipose-derived stromal cells. Outcomes were quite satisfactory¹⁹, but techniques of total fat application are still used^1-3. Furthermore, adipose tissue MSC, when associated with fat grafting, provide greater durability and expressive graft augmentation⁹, and are responsible for the production of collagen in situ, as verified in an animal model by histopathological examination²⁰.

In the present study, adipose tissue MSC were used for the filling of facial rhytids. Mesenchymal stem cells, when associated with hyaluronic acid, are capable of filling deep folds, with a progressive improvement in skin tone and a reduction in fine lines, providing the basis for unique strategies of soft tissue regeneration and correction.

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Publication Dates

• Publication in this collection
  09 Nov 2009
• Date of issue
  Aug 2009

History

• Accepted
  30 Dec 2008
• Received
  28 Oct 2008