All you need to know

Lipofilling of the face

Pr Barbara Hersant MD, PhD.

Pr Barbara HERSANT

Department of plastic, reconstructive, aesthetic and maxillofacial surgery. Henri Mondor Hospital, Créteil, France.

Summary

The advantage of adipose tissue is that it is easy to collect and can be used for autografts. 

The interest of the "lipofilling has long been considered as volumizerbut the discovery of its trophic character has made the lipofilling the most widely used regenerative medicine treatment in the field of tissue regeneration

  • Indeed, adipose tissue is a potential source of adult mesenchymal stem cells (MSCs) with multiple differentiation potentials. 
  • Mesenchymal stem cells (MSCs) are extracted from the cultivation of the vascular stromal fraction (VSF) composed of a heterogeneous population of cells with regenerative properties. 
  • The SVF is derived from enzymatic digestion or mechanical fractionation of adipose tissue. It is thus considered as a alternative cellular product allowing to avoid the culture and cell expansion steps of MSCs.
  • Unfortunately, in France, the use of MSCs and SVF must meet regulatory constraints, being products of cell therapy or "Innovative Therapy Medicines," ITM "(European Directive 2009/12/EC), thus limiting their use by research teams for clinical validation. 
  • These limitations have led to develop new techniques for transferring adipose tissue without going through digestion or cell culture steps. Thus, the development of cannulas allowing the collection of "micro" adipose lobules has allowed the use of a fat rich in stem cells. 
  • More recently, Tonnard et al. have described a mechanical technique allowing to obtain emulsified fat or "nanofat from a sample of microfat. According to the authors, this emulsified fat would be rich in progenitor cells.

These regenerative medicine tools should be used and validated for specific indications in each surgical field.

I/ Lipofilling or fat grafting: an advanced therapy

For a long time, lipofilling was considered to be "volumizing" but the discovery of its trophic character (2000) made lipofilling the most widely used regenerative medicine treatment.

Recent studies have shown that stroma-vascular fraction of adipose tissue represented a reservoir of precursor cells with pro-angiogenic potential was comparable to that of bone marrow-derived stem cells (1). 

In addition, it has been shown that mesenchymal stem cells (CSM) were also present in the adipose tissue. The latter therefore represents a new potential reservoir of pluripotent cells that could be used in regenerative medicine.

Autologous fat transfer is a procedure already applied to obtain the increase of soft tissue volume losses. The fat grafts removed by liposuction are reinjected subcutaneously to restore volume to the defective areas. 

Thus, adipose tissue transplantation is used to correct congenital deformities and complex traumatic wounds with loss of soft tissue, after oncological surgery, after sequelae of radiotherapy... (2).

II/ New techniques for transferring adipose tissue rich in progenitor cells

  • One of the constraints of the use of ITMs in the clinic is the time required and the costs associated with their in vitro amplification.
  • These limitations have led to the development of new cellular products.
  • Several techniques have been proposed for the harvesting of adipose tissue. Coleman et al (3) described a technique that minimizes trauma to the adipocytes. Using a 2-hole, 3-mm cannula with rounded edges connected to a 10-ml syringe, fat is manually aspirated by removing the plunger. The cannula is pushed through the harvest site, the surgeon pulls the plunger of the syringe and creates a slight negative pressure that allows flaps of fat to pass through the cannula and the Luer-Lock opening in the syringe barrel. Once filled, the syringe is disconnected from the cannula, which is replaced with a cap that seals the Luer-Lock end of the syringe. 
  • The grease drawn into the syringes is centrifuged at 3000 RPM (revolutions per minute) for 3 minutes to isolate the grease. 
  • However, reducing the centrifugation time to 1 min has recently been shown to be of value for cell viability (4).
  • After centrifugation, three layers are observed: 
    • The first layer includes oil, which can be removed with an absorbent material, 
    • The second layer is made up of adipose tissue,  
    • And the third layer contains blood, tissue fluid and local anesthetic and is ejected from the base of the syringe. 
    • The middle layer is used for fat grafting (5).
  • New techniques for fat harvesting have been developed using cannulas of only 0.7mm in diameter, used to treat delicate areas of the face such as the eyelids and lips.

III/ Three types of fat are distinguished

1 "Macrofat" fat

Macrofat fat is characterized by lobules of fat with a diameter of 2.4mm.  Macrofat fat is more structural in nature and is easily injected through an 18G or 19G (gauge) cannula. 

Macrofat fat is used for structural augmentation:

  • In the temporal regions, 
  • Deep fat compartments of the cheek:
    • Medial 
    • Prezygomatic, 
    • The piriform region, 
  • The mandible, 
  • The lateral region of the eyebrows, 
  • The nasal bridge and columella 
  • As well as the chin and lips. 

Macrofat fat is at risk for resorption, cystosteatonecrosis, oily cysts, and infection (6-8).

2 " Microfat fat ".

Microfat is characterized by fat lobules with a diameter of 1mm obtained by removing the fat with 2mm diameter cannulas whose multiple holes are each less than 1 mm. 

The micro sampling cannula has ports specifically designed to provide a fat sample consisting of volume-calibrated lobules of adipocytes. 

The deposition microcannulas have a diameter calibrated to the size of the cell units obtained with the sampling cannula. (Figure 1)

Microfat (microfat) is used for trophicity but also for filling.

micro fat
Figure 1: Microlipofilling sampling in a closed circuit to avoid oxidation and contamination of the fat and using cannulas with micro-orifices for sampling.
3 "Nanofat" fat

"Nano-fat or nanofat is characterized by Fat lobules of 400 to 600 μm. Nano-fat is obtained by taking the emulsified micro-fat and passing it between two 10ml syringes linked together by a female-female Luer-Lock connector. After 3 minutes of continuous transfer (20 to 30 passes), the fat has become an emulsified liquid with a whitish appearance rich in SVF.  The emulsified fat is then filtered through a super fine filter to obtain the nanofat (9).

The nanofat can be easily injected with a 27G, 30G, 32G.  It is a cell seeding for the improvement of trophicity.

Nano-fats can be centrifuged to remove free fatty acids and create a gel which can be applied in combination with a cream that promotes dermal penetration and can be applied by mesotherapy techniques, after laser resurfacing or a face lift.

A combination of all three types of fat grafting is used in facial cosmetic surgery or facial fat grafting (10-13).

Tonnard et al have sought to determine the cellular content of nanofat grafts (14). In their study, they showed that the nanofat grafts lacked mature adipocytes and that the native architecture was disrupted. However, the nanografts retained a rich supply of adipose stem cells. Several clinical cases using nanofat grafts have shown an improvement in skin quality 6 months after the procedure. 

Therefore, the author suggests that although nanografts do not contain viable adipocytes, their high stem cell content is clinically useful in skin rejuvenation indications.

IV/ Association of PRP and lipofilling and clinical applications

PRP and adipose tissue are used in different therapeutic areas. (15)

Lipofilling-PRP" in plastic surgery
lookenmedicine

Figure 2: Restoration of breast volume after partial mastectomy by lipofilling.

In plastic, reconstructive and maxillofacial surgery, PRP in combination with adipose tissue is used to :

  • Treat skin lesions, 
  • And for the healing of wounds, especially for the treatment of skin substance losses of origin:
    •  Traumatic, 
    • Post-infectious, 
    • Post-inflammatory
  • The restoration of volumes (Figure 2), 
  • For the treatment of burn scars in order to improve scar trophicity (Figure 3) :
    • Pathological scars (keloid scars), 
    • Skin grafts, 
    • Radiodermatitis 
    • And skin ulcers 
  • This is the case for the filling of pathological defects such as lipodystrophies and facial atrophies. 
  • In maxillofacial and aesthetic surgery, these cellular products are used in 
    • Maxillofacial clefts, 
    • Cervico-facial lifts 
    • And facial rejuvenation. (16-19)
Figure 3: Lipofilling for trophic purposes to treat a deep burn injury.

Use of nanofat and microfat on the face

Nanofat injection in the face could therefore have a stimulating effect on tissue differentiation and regeneration (20). An increase in elastin synthesis and dermal remodeling could be induced by the secretory activity of stem cells mechanically stimulated by the emulsion method (21-23).

Mesguich Batel F. et al (20) reported in a study an improvement of fine lines after treatment with the nanofat method. In addition, the authors characterized the stem cell composition of this method and showed the presence in 1 cm3 of emulsified fat of 23,712 ± 7832 cells/cm3 compared to non-emulsified adipose tissue with a cell viability of 85.1 ± 6.84 % and a proportion of 18.77 ± 6.2 % of regenerating cells.

In addition, intradermal injection of emulsified fat was found to be safe: no erythema, skin discoloration, or inflammatory reactions were seen in all patients (20). (Figure 4 and 5)

Figure 4 : Case 1 : injection of microfat and nanofat with an improvement of the skin trophicity (Case treated by Pr Hersant)
Figure 5: Case 2: injection of microfat-nanofat and botulinum toxin (Case treated by Pr Hersant)

V/ The " Vascular Stroma Fraction " (VSF)

FSV is isolated by digesting the lipid portion of the lipoaspirate with collagenase, separating the contents into two distinct phases: the floating fraction of mature adipocytes and the cellular components of interest in the lower water fraction (24-25). 

  • This separation can be improved by centrifugation, but nevertheless a comparable separation can be achieved by phase separation and gravity filtration (26). 
  • Centrifugation is more efficient because it also allows sedimentation of all cells present, whereas filtration can be designed to capture only important cell types based on their size, thus enriching the specific cell cocktail. 
  • Centrifugation of the aqueous fraction yields a reddish pellet that contains FSV cells. 
  • Erythrocytes, a major contaminant present in the SVF pellet, can be lysed to isolate a purer population of stem cells and/or SVF cells if they are intended for in vitro expansion (27). 
  • The VSF contains a variety of cells: mesenchymal stem cells, pericytes, vascular cells, fibroblasts, pre-adipocytes, monocytes, macrophages, red blood cells, fibrous tissue and extracellular matrix (ECM). 

For clinical applications in regenerative medicine, one of the advantages of using FSV is its extemporaneous use without going through the cell culture steps.

  • The number of stem cells contained in adipose SVF can fluctuate considerably as patients may have different adipose tissue texture and density (28).

VI/ Adipose tissue stem cells

Adipose Derived Stem Cells (ADSC) were first characterized in 2001 and have since been widely studied and used as a major source of cells with regenerative potential, with characteristics similar to those of mesenchymal stem cells (MSCs) (29-31). Stem cells are cells capable of self-renewal, generating identical daughter cells to maintain the stem cell pool, and differentiating into multiple cell lines (progenitors with a more restricted potential). 

        Characterization of adipose tissue MSCs (differentiation potential, proliferation, paracrine and immunomodulatory actions)

  • Several studies have demonstrated the ability of adipose tissue stem cells to differentiate into adipogenic, osteogenic and chondrogenic lineages, and even into myogenic lineages, leading to skeletal muscle, smooth muscle and cardiomyocytes (32). 
  • MSCs have also been shown to possess the potential to differentiate into cells of both ectodermal and endodermal lineages, such as neuronal cells, endothelial cells, epithelial cells, hepatocytes, pancreatic cells, and hematopoietic cells (33-39). 
  • In addition, they also have a migration potential (40) and a capacity to proliferate that can be stimulated by various growth factors. (41). 
  • The main effects of MSCs are mediated by paracrine activity. ASCs produce and secrete a wide variety of growth factors, cytokines, and chemokines that may be involved in wound healing by secreting angiogenic and anti-apoptotic growth factors (42). 
  • In addition, adipose tissue MSCs modulate monocytes, macrophages, T and B lymphocytes by inducing the host immune response.
  • MSCs from adipose tissue in cell therapy
  • One of the advantages of using MSCs from adipose tissue, and not the least, is the ease of obtaining these cells. They do not pose any ethical problems, unlike embryonic stem cells. 
  • Indeed, they are recovered directly from the fat of the "surgical waste", after dermolipectomy or liposuction. 10 to 100 million stem cells can be obtained from 300 milliliters of lipoaspirate of which more than 90% would be viable (43). A large number of stem cells can be obtained in a few passages.
  • Optimization of cell therapy protocols using adipose tissue MSCs
  • Regenerative medicine, which aims to stimulate tissue repair and regeneration mechanisms, offers the possibility of effective treatment strategies in combination. 
  • However, in this field, the tissue engineering approaches proposed so far have involved in vitro expansion of autologous or allogeneic cells (44). 
  • The future of reconstructive surgery will favor autologous cell therapy, if possible extemporaneous, for skin regeneration. 
  • Despite promising results in animal models of injury, the use of ASCs (Adipose Stem Cells)  has shown mixed results in clinical trials for skin healing (45), due to their poor viability after transplantation.
  • In a study published in "International Stem Cells", we showed that the combination of PRP with cell therapy by injection of adipose tissue MSCs seems to improve skin healing in the mouse model. 
  • Indeed, treatment with MSC injections and the combination of MSC and PRP significantly improves skin regeneration. 
  • Time to healing was also significantly improved by the combination of MSC and PRP compared to MSC alone, PRP alone and the control. 
  • We were able to explain these preclinical results by the demonstration of activation of the MSC secretome by PRP, reflected by activation of human-derived VEGF and IL-6 transcriptional expression in mouse transplant biopsies and also by increased MSC survival (46).

VII/ Bioethics law and regulatory aspects

Blood derivatives: precise regulations

Autologous Platelet Concentrate (APC) preparations are considered human blood products. 

      • They are therefore subject to Article L. 1221-8 of the Public Health Code. 
      • In contrast, the Public Health Code provides for the use of autologous platelet concentrates for therapeutic purposes in the context of a single medical intervention, without being stored or prepared within a third-party organization or institution.
      • In a press release published on January 10, 2018, the ANSM (National Agency for the Safety of Medicines and Health Products) reminds that the cosmetic use of autologous platelet concentrates (CPA), also known as platelet-rich plasma (PRP), is prohibited in France. 
      • It is therefore the objective of the intervention (therapeutic or aesthetic) that determines the applicable regulations. 
      • It is therefore possible to use PRP in functional or reconstructive surgery (e.g. scarring).

The use of stem cells is qualified by the ANSM as "cellular products for therapeutic purposes" are human cells used for autologous therapeutic purposes, 

  • When these cellular products for therapeutic purposes are pharmaceutical specialties or other industrially manufactured medicines (advanced therapy medicinal product - ITM), they are governed by the rules applicable to medicines. 
  • They also fall within the scope of pharmacovigilance.  

ITMs are cells or tissues that have been subjected to substantial manipulation so as to obtain biological characteristics, physiological functions, or structural properties useful for the intended regeneration, repair, or replacement and/or the cells or tissues are not intended to be used for the same essential function(s) in the recipient and the donor. 

  • According to the regulation 1394/2007, the non-substantial manipulations include: cutting, crushing, centrifugation, sterilization, irradiation, separation, concentration, cryopreservation and freezing.
  • Four categories of ITMs are defined: gene therapy ITMs, somatic cell therapy ITMs, tissue and cell engineering ITMs and combined advanced therapy drugs (combining an ITM with a medical device). 
  • When they are not industrially manufactured, they are then qualified as cell therapy preparations, including when human cells are used to transfer genetic material. They are thus subject to biovigilance.
  • Tocell therapy and bioethics law

Industrially manufactured cellular products for therapeutic purposes are subject to the requirement, prior to their commercialization, to obtain a marketing authorization. 

    • They must be manufactured in licensed pharmaceutical facilities.
    • Cell therapy preparations, which are not manufactured industrially, are subject to prior authorization by the ANSM, which obtains the opinion of the Agence de biomédecine beforehand. 
    • This authorization is issued after evaluation of their preparation and conservation process and their therapeutic indications.
    • The ANSM also issues authorizations to establishments or organizations carrying out the activities of preparation, storage, distribution and transfer, for autologous or allogeneic therapeutic purposes, of cell therapy preparations. The authorization is issued after the opinion of the biomedicine agency (47).
    • Non-substantial handling according to Regulation (EC) 1394/2007 :
        1. Cutting
        2. Grinding
        3. Centrifugation
        4. Sterilization
        5. Irradiation
        6. Separation, concentration
        7. Cryopreservation, freezing

Conclusion

  • Emulsified fat thus seems to be a simple alternative to cell therapy procedures and SVF preparation.
  • The injection of fragmented adipocytes present in emulsified fat and the cytokines released could therefore have a stimulating effect on tissue differentiation and regeneration.
  • In the field of regenerative medicine, locellular biotherapies offer major perspectives in many pathologies where therapeutic resources are currently insufficient.
  • Moreover, the development of these cellular products is now based on progress in the fundamental knowledge of the physiopathological processes underlying the disease in question, as well as on the precise mechanism of action of the cells administered.

References

  • Zimmerlin L, Donnenberg VS, Pfeifer ME, Meyer EM, et al. Stromal vascular progenitors in adult human adipose tissue. Cytometry A 2010; 77:22-30.
  • Simonacci F, Bertozzi N, Grieco MP, Grignaffini E, Raposio E. Procedure, applications, and outcomes of autologous fat grafting. Ann Med Surg (Lond). 2017 Jun 27; 20:49-60.
  1. Coleman S.R. Structural fat grafts: the ideal filler? Clin Plast Surg 200;28(1):111-119.
  2. Hoareau L, Bencharif K, Girard AC, Gence L, Delarue P, Hulard O, Festy F, Roche R. Effect of centrifugation and washing on adipose graft viability: a new method to improve graft efficiency. J Plast Reconstr Aesthet Surg. 2013 May;66(5):712-9.
  3. Coleman SR. Structural fat grafting: more than a permanent load. Plast. Reconstr. Surg. 2006; 118: 108S-120.
  4. Eto H., Kato H., Suga H., Aoi N., Doi K., Kuno S., and Yoshimura K. The fate of adipocytes after nonvascularized fat grafting: evidence of early death and replacement of adipocytes. Plast Reconstr Surg 2012; 129:1081.
  5.  Alexander D, Bucky LP. Breast augmentation using preexpansion and autologous fat transplantation- a clinical radiological study. Plast Reconstr Surg 2011; 127:2451-2452.
  6. Spear SL, Pittman T. A prospective study on lipoaugmentation of the breast. Aesthet Surg J. 2014 Mar;34(3):400-8.
  7. Wei H, Gu SX, Liang YD, Liang ZJ, Chen H, Zhu MG, Xu FT, He N, Wei XJ, Li HM. Nanofatderived stem cells with platelet-rich fibrin improve facial contour remodeling and skin rejuvenation after autologous structural fat transplantation. Oncotarget. 2017 Jul 31;8(40):68542-68556.
  8. Cohen SR, Hewett S, Ross L, Delaunay F, Goodacre A, Ramos C, Leong T, Saad A. Regenerative Cells For Facial Surgery: Biofilling and Biocontouring. Aesthet Surg J. 2017 Jul 1;37(suppl_3):S16-S32.
  9. Dasiou-Plakida D. Large injections for facial rejuvenation: 17 years of experience in 1720 patients. J. Cosmet. Dermatol. 2003; 2: 119-125.
  10. Mazzola RF Quality Medical Edition; St. Louis, MO: 2009. Fat injection: from filling to regeneration; pp. 373-422.
  11. Tonnard P, Verpaele A, Peeters G, Hamdi M, Cornelissen M, Declercq H. Nanofat grafting: basic research and clinical applications. Plast Reconstr Surg 2013; 132:1017-26.
  12. Chou TM, Chang HP, Wang JC. Autologous platelet concentrates in maxillofacial regenerative therapy. Kaohsiung J Med Sci. 2020 Feb 12.
  13. Picard F, Hersant B, Bosc R, Meningaud JP. The growing evidence for the use of platelet-rich plasma on diabetic chronic wounds: A review and a proposal for a new standard care. Wound Repair Regen. 2015 Sep;23(5):638-43
  14. Picard F, Hersant B, Bosc R, Meningaud JP. Should we use platelet-rich plasma as an adjunct therapy to treat "acute wounds," "burns," and "laser therapies": A review and a proposal of a quality criteria checklist for further studies. Wound Repair Regen. 2015 Mar-Apr;23(2):163-70.
  15. Hersant B., SidAhmed-Mezi M., Bosc R., Meningaud J.P. Autologous Platelet-Rich Plasma/Thrombin Gel Combined with Split-Thickness Skin Graft to Manage Postinfectious Skin Defects: A Randomized Controlled Study. Adv. Skin Wound Care. 2017; 30:502-508. 
  16. Hersant B, SidAhmed-Mezi M, Picard F, Hermeziu O, Rodriguez AM, Ezzedine K, Meningaud JP. Efficacy of Autologous Platelet Concentrates as Adjuvant Therapy to Surgical Excision in the Treatment of Keloid Scars Refractory to Conventional Treatments: A Pilot Prospective Study. Ann Plast Surg. 2018 Aug;81(2):170-175.
  17. Davis NF, Cunnane EM, Quinlan MR, Mulvihill JJ, Lawrentschuk N, Bolton DM, Walsh MT. Biomaterials and Regenerative Medicine in Urology. Adv Exp Med Biol.2018; 1107:189-198.
  18. Dawood AS, Salem HA. Current clinical applications of platelet-rich plasma in various gynecological disorders: An appraisal of theory and practice. Clin Exp Reprod Med. 2018 Jun;45(2):67-74.
  19. Jones IA, Togashi RC, Thomas Vangsness C Jr. The Economics and Regulation of PRP in the Evolving Field of Orthopedic Biologics. Curr Rev Musculoskelet Med. 2018 Dec;11(4):558-565.
  20. Yoshimura K., Sato K., Aoi N., Kurita M., Hirohi T., and Harii K. Cell-assisted lipotransfer for cosmetic breast augmentation: supportive use of adipose-derived stem/stromal cells. Aesthetic Plast Surg 2008; 32:56-57.
  21. Yoshimura K., Sato K., Aoi N., Kurita M., Inoue K., Suga H., Eto H., Kato H., Hirohi T., and Harii K. Cell-assisted lipotransfer for facial lipoatrophy: efficacy of clinical use of adiposederived stem cells. Dermatol Surg 2008; 34:1178.
  22. Chung C.W., Marra K.G., Li H., Leung A.S., Ward D.H., Tan H., Kelmendi-Doko A., and Rubin J.P. VEGF microsphere technology to enhance vascularization in fat grafting. Ann Plast Surg 69,213, 2012. 168
  23.  Lu F., Li J., Gao J., Ogawa R., Ou C., Yang B., and Fu B. Improvement of the survival of human autologous fat transplantation by using VEGF-transfected adipose-derived stem cells. Plast Reconstr Surg 2009; 124:1437.
  24. Yuksel E., Weinfeld A.B., Cleek R., Wamsley S., Jensen J., Boutros S., Waugh J.M., Shenaq S.M., and Spira M. Increased free fat-graft survival with the long-term, local delivery of insulin, insulin-like growth factor-I, and basic fibroblast growth factor by PLGA/PEG microspheres. Plast Reconstr Surg 2000; 105:1712.
  25. Hamed S., Egozi D., Kruchevsky D., Teot L., Gilhar A., and Ullmann Y. Erythropoietin improves the survival of fat tissue after its transplantation in nude mice. PLoS One 2010;5:13986.
  26. Marx RE. Platelet-rich plasma (PRP): what is PRP and what is not PRP? Implant Dent. 2001; 10:225-228.
  27. Anitua E., Alkhraisat M.H., Orive G. Perspectives and challenges in regenerative medicine using plasma rich in growth factors. J. Control. Release. 2012; 157:29-38.
  28. Dohan Ehrenfest DM, Rasmusson L, Albrektsson T. Classification of platelet concentrates: from pure platelet-rich plasma (P-PRP) to leukocyte-and platelet-rich fibrin (L-PRF) Trends Biotechnol. 2009; 27:158-167.
  29. Weibrich G, Kleis WK, Hafner G, Hitzler WE, Wagner W. Comparison of platelet, leukocyte, and growth factor levels in point-of-care platelet-enriched plasma, prepared using a modified Curasan kit, with preparations received from a local blood bank. Clin Oral Implants Res. 2003; 14:357-162.
  30. Gonshor A. Technique for producing platelet-rich plasma and platelet concentrate: background and process. Int J Periodontics Restorative Dent. 2002;22:547-557. 
  31. Roubelakis M.G., Trohatou O., Roubelakis A., Mili E., Kalaitzopoulos I., Papazoglou G., Pappa K.I., Anagnou N.P. Platelet-rich plasma (PRP) promotes fetal mesenchymal stem/stromal cell migration and wound healing process. Stem Cell Rev. 2014; 10:417-428.
  32. Cross K.J., Mustoe T.A. Growth factors in wound healing. Surg. Clin. N. Am. 2003; 83:531-545.
  33. Demidova-Rice T.N., Hamblin M.R., Herman I.M. Acute and impaired wound healing: Pathophysiology and current methods for drug delivery, part 2: Role of growth factors in normal and pathological wound healing: Therapeutic potential and methods of delivery. Adv. Skin Wound Care. 2012; 25:349-370.
  34. Hersant B, Bouhassira J, SidAhmed-Mezi M, Vidal L, Keophiphath M, Chheangsun B, Niddam J, Bosc R, Nezet AL, Meningaud JP, Rodriguez AM. Should platelet-rich plasma be activated in fat grafts? An animal study. J Plast Reconstr Aesthet Surg. 2018 May;71(5):681-690.
  35. Matsumoto D, Sato K, Gonda K, et al. Cell-assisted lipotransfer: supportive use of human adipose-derived cells for soft tissue augmentation with lipoinjection. Tissue Eng. 2006; 12:3375-82.
  36. Zuk PA, Zhu M, Mizuno H, et al. Multilineage cells from human adipose tissue: implications for cell-based therapies. Tissue Eng. 2001; 7:211-28.
  37. SundarRaj S, Deshmukh A, Priya N, et al. Development of a system and method for automated isolation of stromal vascular fraction from adipose tissue lipoaspirate. Stem Cells Int. 2015; 2015:1-11.
  38. Riis S, Zachar V, Boucher S, et al. Critical steps in the isolation and expansion of adiposederived stem cells for translational therapy. Expert Rev Mol Med. 2015; 17:11.
  39. Martin AD, Daniel MZ, Drinkwater DT, Clarys JP. Adipose tissue density, estimated adipose lipid fraction and whole-body adiposity in male cadavers. Int J Obes Relat Metab Disord. 1994;18(2):79-83.
  40. Bourin P, Bunnell BA, Casteilla L, Dominici M, Katz AJ, March KL, Redl H, Rubin JP, Yoshimura K, Gimble JM. Stromal cells from the adipose tissue-derived stromal vascular fraction and culture expanded adipose tissue-derived stromal/stem cells: a joint statement of the International Federation for Adipose Therapeutics and Science (IFATS) and the International Society for Cellular Therapy (ISCT) Cytotherapy. 2013; 15:641-8.
  41. Gimble JM, Bunnell BA, Frazier T, Rowan B, Shah F, Thomas-Porch C, Wu X. Adiposederived stromal/stem cells: a primer. Organogenesis. 2013 Jan-Mar;9(1):3-10.
  42. Nguyen A, Guo J, Banyard DA, Fadavi D, Toranto JD, Wirth GA, Paydar KZ, Evans GR, Widgerow AD.Stromal vascular fraction: a regenerative reality? Part 1: current concepts and review of the literature. J Plast Reconstr Aesthetic Surg. 2016; 69:170-9. 
  43. Bunnell BA, Flaat M, Gagliardi C, Patel B, Ripoll C. Adipose-derived stem cells: isolation, expansion and differentiation. Methods. 2008 Jun;45(2):115-20.
  44. Fraser J.K., Schreiber R., Strem B. Plasticity of human adipose stem cells toward endothelial cells and cardiomyocytes. Nat. Clin. Pract. Cardiovasc Med. 2006;3: S33-S37.
  45. Fujimura J., Ogawa R., Mizuno H., Fukunaga Y., Suzuki H. Neural differentiation of adipose-derived stem cells isolated from GFP transgenic mice. Biochem. Biophys. Res. Commun. 2005; 333:116-121.
  46. Planat-Benard V., Silvestre J.S., Cousin B. Plasticity of human adipose lineage cells toward endothelial cells: physiological and therapeutic perspectives. Circulation. 2004; 109:656-663.
  47. Brzoska M., Geiger H., Gauer S., Baer P. Epithelial differentiation of human adipose tissuederived adult stem cells. Biochem. Biophys. Res. Commun. 2005; 330:142-150.
  48. Banas A., Teratani T., Yamamoto Y. Adipose tissue-derived mesenchymal stem cells as a source of human hepatocytes. Hepatology. 2007; 46:219-228.
  49. Timper K., Seboek D., Eberhardt M. Human adipose tissue-derived mesenchymal stem cells differentiate into insulin, somatostatin, and glucagon expressing cells. Biochem. Biophys. Res. Commun. 2006; 341:1135-1140.
  50. Corre J., Barreau C., Cousin B. Human subcutaneous adipose cells support complete differentiation but not self-renewal of hematopoietic progenitors. J. Cell Physiol. 2006; 208:282-288.
  51. Scanarotti C., Bassi A.M., Catalano M. Neurogenic-committed human pre-adipocytes express CYP1A isoforms. Chem. Biol. Interact. 2010; 184:474-483.
  52. Coradeghini R., Guida C., Scanarotti C. A comparative study of proliferation and hepatic differentiation of human adipose-derived stem cells. Cells Tissues Organs. 2010; 191:466-477.
  53. Aluigi M.G., Coradeghini R., Guida C. Pre-adipocyte commitment to neurogenesis 1: preliminary localization of cholinergic molecules. Cell Biol. Int. 2009; 33:594-601.
  54. Izadpanah R., Kaushal D., Kriedt C. Long-term in vitro expansion alters the biology of adult mesenchymal stem cells. Cancer Res. 2008; 68:4229-4238.
  55. Mizuno H, Tobita M, Uysal AC. Concise Review: Adipose-Derived Stem Cells as a Novel Tool for Future Regenerative Medicine. Stem cells. 2012; 30:804-810.
  56. Rehman J, Traktuev D, Li J, et al. Secretion of angiogenic and antiapoptotic factors by human adipose stromal cells. Circulation 2004; 109:1292-8.
  57. Locke M, Windsor J, Dunbar PR. Human adipose-derived stem cells: isolation, characterization and applications in surgery. ANZ J Surg. 2009; 79: 235-244.
  58. Böttcher-Haberzeth S, Biedermann T, Reichmann E. Tissue engineering of skin. Burns. 2010 Jun;36(4):450-60.
  59. Sorrell JM, Caplan AI. Topical delivery of mesenchymal stem cells and their function in wounds. Stem Cell Res Ther. 2010; 1:30.
  60. Hersant B, Sid-Ahmed M, Braud L, Jourdan M, Baba-Amer Y, Meningaud JP, Rodriguez AM. Platelet-Rich Plasma Improves the Wound Healing Potential of Mesenchymal Stem Cells through Paracrine and Metabolism Alterations. Stem Cells Int. 2019 Oct 31; 2019:1234263.
  61. Boucher, H., & Cras, A. (2018). Advanced therapy drugs: regulation and clinical applications. Revue Francophone Des Laboratoires, 2018(507), 44-51.