Fat Grafting for Rejuvenation and Regeneration with Stromal Vascular Fraction Gel

Wenqing JIANG ,Yunjun LIAO,Feng LU

1.2 Department of Plastic and Cosmetic Surgery,Nanfang Hospital,Southern Medical University,Guangzhou 510515,China.

SUMMARY Lipotransfer has become a powerful regenerative tool,largely because of its cellular components,the stromal vascular fraction (SVF).However,the clinical separation of cells with collagenase is strictly legislated.In 2017,Yao et al.postulated a novel fat-derived product mechanically concentrating SVF cells and an extracellular matrix (ECM) and named it stromal vascular fraction gel (SVF-gel).This review discussed the protocol of SVF-gel and its component as well as its inner structure.The histologic examination and the retention rate after the transplantation of SVF-gel were also rendered.Moreover,we summed up the rejuvenating and regenerative use of SVF-gel and introduced its possible mechanism.

KEY WORDS Fat grafting; Stromal vascular fraction; Rejuvenation; Regeneration; Cell-based therapy

INTRODUCTION

Lipotransfer is a well-established surgical technique for volumetric use.Several previous studies conducted since 2006 regarding lipotransfer have focused on tissue regenerative properties[1],which correlate with lipoaspirate[2].Lipoaspirate contains adipocytes and stromal vascular fraction (SVF) cells,including several cell populations,especially adipose-derived stem cells(ASCs)[3-5].Compared to other adult stem cells,ASCs are easily accessible and possess feasibility for tissue repair as well as tissue regeneration with their capacity to secrete angiogenic and immunomodulatory factors and to proliferate and differentiate into different cell types[6-9].Therefore,interest in ASCs has dramatically increased worldwide for reconstructive and esthetic purposes,and clinical trials of ASCs have increased exponentially[10-11].

However,limitations for clinical application exist,especially for immediate application after liposuction,since the separation of ASCs requires complicated procedures and mostly introduces foreign collagenase during processing,increasing the risk of exogenous biological contamination[12-15].Then,the purification and cultivation of ASCs require special devices[16-17].Moreover,the separated ASC suspensions are often applied solely without the protection of the ECM components,rendering the transplanted cells vulnerable to recognition and eradication by the immune system[18-21].Therefore,to maximize regenerative use,it is essential to seek a safe,efficient,and reliable method of obtaining ASCs from lipoaspirate.

In 2017,Yao et al.proposed a new adipose-derived product,SVF-gel,which is an injectable mixture generated from lipoaspirate through a simple mechanical process[22].As a cytotherapy tool based on SVF cells and ASCs,SVFgel is prepared using a minimally invasive procedure and in the absence of collagenase digestion.The whole process can be effectively achieved in less than 2 h,enabling simple high availability of ASCs in operations.Moreover,the concentration of ASCs in SVF-gel is markedly increased compared to that in Coleman fat[23-24].

Therefore,the advantages of harboring abundant SVF cells with no requirement for separation and little preparation time make SVF-gel a reliable therapeutic approach for rejuvenation and regeneration.

Fat Harvesting and SVF-Gel Preparation

Under the tumescent anesthesia technique,human lipoaspirate is usually obtained from the abdomen and inner thigh.A 20 mL Luer-Lok syringe or vacuum suction is connected to a 3 mmol/L multi-hole cannula,consisting of incisive 1 mmol/L-diameter lateral holes for harvesting.Liposuction is performed at -0.75 atm pressure.

For sedimentation for 10 min,the middle layer of lipoaspirate is collected for the first centrifugation at 1,200 × g for 3 min to obtain Coleman fat.Then,the liquid portion is discarded.The Coleman fat is transferred into two 10 mL syringes connected by a Luer-Lok connector(internal diameter of 2.4 mmol/L) and mechanically emulsified by transferring between two syringes (six to eight times) at a rate of 20 mL/s until the Coleman fat converted into a uniform emulsion[25-27].Shifting the fat back and forth between syringes generates a shear force to break the adipose cells,which are more vulnerable than other cellular components because of their enormous volume and delicate structure.The emulsion is then centrifuged at 2,000 × g for 3 min.Finally,only the middle sticky substance is defined as SVF-gel,while the rest is discarded.

As Yao et al.defined in 2017[22]:

Condensing rate=(SVF-gel volume)/ [(oil volume) +(liquid volume)+(SVF-gel volume)]

The enriched SVF-gel as a reliable cytotherapy tool is defined as follows:

1.A final volume less than 15% of the initial volume;

2.An SVF cell density greater than 4.0 × 105cells/mL;

3.A product injectable through a 27-gauge needle.

Cellular Component and Extracellular Matrix of SVFGel

The emulsification process makes the SVF-gel have a smooth liquid-like texture.Although the shear force may cause damage to some SVF cells,the SVF-gel still contains a high density of functional cell populations in lipoaspirate.The concentration of SVF cells in SVF-gel reaches (2.7 ± 0.3) × 105cells/mL[28].Flow cytometry has identified two main cell subpopulations in SVF-gel; ASCs(CD34+/CD31-/CD45-,64% of SVF cells) and endothelial cells (ECs,CD34+/CD31+/CD45-,28% of SVF cells)[23].Their concentrations in SVF-gel are (1.9 ± 0.2) × 105cells/mL and (7.7 ± 2.4) × 104cells/mL,respectively[22].ASCs isolated from SVF-gel can proliferate and have been induced to adipogenic,osteogenic,and chondrogenic differentiation[24,29].

Scanning electron microscopy was previously performed to observe the ECM structure of the SVF-gel.Normal fat has a relatively well-integrated ECM ultrastructure with adipose cells adhering to extracellular fibers[22,24].In contrast,few adipocytes exist within the SVF-gel,but a large number of ECM components after the mechanical process are not statistically significant.In terms of ECM integrity,there are differences between SVF-gel and fresh adipose tissues.Compared with the SVF suspension,the ECM scaffold in the SVF-gel accommodates viable ASCs within their natural niche,ensuring that ASCs and other adhering cells maintain optimal cell elasticity and migration and adipose re-growing[30-31].Specifically,the elasticity,nanotopography,and protein content of ECM are crucial factors regulating the behavior of ASCs[32-33].As native ECM retains many growth factors,it not only serves as a structural scaffold but may also have chemoattractant effects on vascular ingrowth and adipogenesis[34-36].

Histologic Evaluation and Retention of SVF-Gel Grafts

The effect on tissue volumization of SVF-gel was previously assessed in an experimental study[37].A significant difference was identified between the SVFgel and Coleman Fat in terms of long-term retention volume (P＜ 0.05).SVF-gel was 80% ± 15% of the initial injection volume after transplantation for three months versus 42% ± 9% following Coleman fat graft.The volume of Coleman fat decreased remarkably from 2 weeks with poor neovascularization and visible lipid droplets within adipocytes,while the volume remained stable in the SVF-gel group and the inner texture of the grafts developed similarly to normal adipose tissue at each time point.Although it showed a decreasing tendency for the first 3 days,the retention rate rebounded to the initial volume on day 11 and remained constant thereafter.

A unique adipose regeneration mode may be involved in the mechanism of the improved retention of SVFgel,involving immediate inflammation and soakage of inflammatory cells,activating instant vascularization,and initiating host-mediated adipose regeneration[38-40].For the first 3 days after transplantation of SVF-gel,many preadipocytes are characterized by small size and multiple intracellular lipid droplets,and well-developed vasculature and inflammatory infiltration have been observed in both the superficial and central areas of SVF-gel grafts.Consistent with the volume change,the number of immune cells begins to diminish,and most of the macrophages are M2 polarized (Mac+/CD206+).It is fully replaced by mature adipocytes with greater integrity until day 90.The level of fibrosis is surprisingly low in the SVF-gel group on days 30,60,and 90 (P＜ 0.05).In addition,in terms of the histologic origin of newlyformed adipocytes,they are almost all host-derived.

Volumization and Rejuvenation Use of SVF-Gel

The high retention rate assumes that SVF-gel is an inspiring filler for tissue volumization and rejuvenation.A retrospective,single-center study compared patient satisfaction and secondary surgery rates between patients who received SVF-gel injection and those who underwent conventional fat grafting[41].Improvements in facial augmentation and contour were observed in all patients.A 5-point Likert scale assessing patients’ satisfaction showed that 54.5% of the patients in the SVF-gel group were satisfied with their outcomes and 22.8% were very satisfied,while the rates were 48.7% and 5.1%,respectively,in the conventional fat grafting group.The secondary surgery rate was significantly lower in the SVF-gel group than in the conventional fat grafting group (P＜ 0.001).

Traditional lipoinjection is always administered through an 18-gauge needle to ensure that large particles,such as mature adipocytes,can be reinjected successfully into the subcutaneous layer.The heavy gauge makes it difficult for precise facial contouring,which can lead to a bumpy or dimpled skin surface and noticeable nodules.Comparatively,SVF-gel can be easily injected through a fine,27-gauge needle with a smooth liquid-like texture[22].This enables flexible filling of SVF-gel injection and is less likely to appear as uneven structures.The minimally invasive process also means attenuated inflammation without severe pain and swelling post-injection,allowing for a rapid recovery period for the patients.The participants in the SVF-gel group developed mild postoperative swelling or no swelling,which disappeared in 1 to 2 weeks[41].

SVF-gel also exhibits a satisfactory anti-aging effect for horizontal neck lines,eye bags,apparent deformity in the lacrimal groove,and maxillary retrusion[25-26].Meanwhile,most patients in the reported cases showed an improvement in skin texture after SVF-gel injection.

SVF-Gel and Wound Healing

SVF-gel has a great regenerative function,especially in wound healing.A STROBE-compliant study showed a great therapeutic outcome of SVF-gel on chronic wounds unhealed for more than three months[42].The SVF-gel group exhibited a significantly lower rate of 34.55% ± 11.18% than that of the negative pressure wound therapy group,which had a wound healing rate per week of 10.16% ± 2.67% (P＜ 0.001).In deep dermis layers,inflammatory cell infiltration was alleviated after applying SVF-gel for 2 weeks along with increased collagen deposition and neovascularization in the recipient sites.The treatment outcome of SVF-gel has also been evaluated in a nude mouse excisional wound healing model,taking an SVF cell group and PBS group as controls[22,24,28].On day 14 post-transplantation,the SVF-gel group healed completely,showing a notably rapid wound healing rate,while control wounds remained unclosed (P＜ 0.05).Macroscopic visualization revealed that subcutaneous blood vessels and their small branches around the wound extended into the SVF-gel graft,developing a vascular network to supply nutrition for wound healing.Moreover,another study established that subcutaneous SVF-gel application increased the survival area of the free flap by accelerating flap neovascularization[29].

The underlying wound healing mechanism of SVFgel can be speculated from three factors.The first factor may be the rapid inflammatory response soon after transplantation.As shown in the murine wound healing model,a significantly higher expression of the inflammatory cytokine monocyte chemoattractant protein (MCP)-1 and prominent soakage of inflammatory cells were observed in the SVF-gel group in the early stages.The levels of inflammation decreased remarkably in the later period[24].In response to inflammatory chemoattractants,large amounts of monocytes infiltrate the wound and differentiate into activated macrophages,secreting high concentrations of cell factors,such as platelet-derived growth factor,vascular endothelial growth factor (VEGF),and basic fibroblast growth factor(bFGF).During the tissue reconstruction phase,the attenuated inflammation level,serving as a protection mechanism,helps prevent scar formation and improves prognosis in the wound area[43].

Except for the proangiogenic stimulus of inflammation,the SVF-gel SVF cells,especially ASCs,can promote reepithelization,neovascularization,collagen metabolism,and immunomodulation through secretion of many growth factors,such as VEGF,hepatocyte growth factor,epidermal growth factor,and bFGF[44-45].The capillary density was evaluated in the murine wound healing model after SVF-gel treatment and was higher than that in the SVF cell group from day 4 to 14 (P＜ 0.05),with an increased expression of the angiogenic factors VEGF and bFGF (P＜ 0.05)[24].In addition,the ability to differentiate into epidermal cells and ECs makes ASCs suitable for treating chronic wounds with SVF-gel as it induces re-epithelization and angiogenesis[3,46].ECs constitute capillaries directly and are critical elements in neovascularization.HUVECs cultured in SVF-gel extracts for 24 hours form numerous interconnected tubular structures,while those cultivated in a mixture of VEGF and bFGF and a normal culture medium fail to form an anastomotic vascular network[24].

Another factor that facilitates wound healing is the ECM,which contains fibronectin,glycosaminoglycans,elastin,and growth factors[35].ECM may play a crucial role in vascular cell migration and vasculature morphogenesis,since it provides a collagen framework during the wound healing process[32,36].In addition to structural support,the synergistic interaction between SVF cells and ECM components in SVF-gel seems to be an essential factor in promoting wound healing.In principle,the native ECM within SVF-gel provides a favorable cellular microenvironment for SVF cells to survive and proliferate,facilitating the wound healing process[30].

SVF-Gel and Fibrotic Conditions

As the key component in SVF-gel,ASCs have antifibrotic and regenerative properties in many fibrotic conditions,which makes SVF-gel an ideal treatment for fibrotic disease.In a hypertrophic scar model,the scar significantly softened and contracted 4 weeks after SVFgel injection and became almost unnoticeable after 12 weeks with an intact epidermal covering and a thinner dermal layer,restoring a firm subcutaneous layer with substantial adipose tissue[23].In three reported cases of skin necrosis related to the nasal injection of hyaluronic acid,patients who developed hypertrophic scars in either nasal tip,nasal apex,or nasal alar received SVFgel treatment[27].After several SVF-gel injections,the color,texture,elasticity,and softness of the scars were significantly improved and the volume of the nasal apex was restored while pain and itching were gradually relieved.One of the patients underwent biopsies before and after injection.These showed that the collagen density within the scar decreased and mature adipocytes and well-developed vessels appeared.

Experimental and clinical evidence suggests the potential mechanisms of SVF-gel in fibrotic conditions.The mRNA expression of inflammatory cytokines MCP-1 and interleukin (IL)-6 is surprisingly lower in a SVF-gel group (P＜ 0.05) in parallel with alleviated soakage of macrophages (CD206+) in the dermal connective tissue[23].Compared with SVF cells and PBS-treated groups,the collagen density is much lower in a SVF-gel group and the quantitative myofibroblast infiltration is the lowest among all groups 12 weeks after transplantation (P＜ 0.05),as is the deposition of collagen I (Col-1) at week 4 (P＜ 0.05).

SVF-gel relieves macrophage-mediated inflammation and prevents excessive accumulation of ECM proteins,such as Col-1,through the activities of myofibroblasts,inhibiting the formation and development of hyperfibrosis of the skin[47-49].ASCs suppress the production of proinflammatory cytokines in certain activated macrophages and induce their apoptosis,conferring resistance to M1-polarization gene expression in macrophages,which is characterized by low CD206 expression[50-53].

CONCLUSION

SVF-gel is an autologous injectable derived from the native ECM and is a functional cellular component generated using a simple mechanical process.It offers a novel mode of tissue rejuvenation and regeneration suitable for clinical applications in stem cell therapies.

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Competing Interests

The authors declare that they have no competing interests.The authors state that the views expressed in the submitted article are their own and not the official position of the institution or funder.