Synergistic effect of nanofat and mouse nerve‐growth factor for promotion of sensory recovery in anterolateral thigh free flaps

Abstract Anterolateral thigh (ALT) free flaps are widely used for reconstruction, but poor sensory recovery of the flap tissue can cause unsatisfactory outcomes and poor function. Adipose‐derived mesenchymal stem cells (ADSCs) promote neural regeneration, but the clinical use of stem‐cell therapy has been limited by lack of regulatory approval. Nanofat is an autologous product that is prepared mechanically from harvested fat. It is enriched in ADSCs and does not contain any exogenous substances. The developmental and adult neurobiology of nerve‐growth factor (NGF) are well investigated, and mouse (m)NGF has been used to promote recovery following peripheral nerve injury. We investigated the promotion of nanofat and mNGF as either mono‐ or combined therapy on the sensory recovery of ALT free flaps. We found that nanofat and mNGF had a synergistic effect on sensory recovery that was associated with stimulation of angiogenesis and neurogenesis. Nanofat combined with mNGF was better at promoting neural regeneration and improving sensory recovery than treatment with either agent alone. The results provide a theoretical rationale for further study of the clinical use of nanofat combined with mNGF to promote the sensory recovery of ALT free flaps.

sensory nerve fibers in the dermis or epidermis may result in poor sensory recovery. The harvesting of ALT free flaps with donor nerves and subsequent microsurgical anastomosis with the recipient nerves promotes sensory recovery. 8,9 However, numbness and sensory disorders may occur because of the denervation of the donor site, and neuromas that occur at sites of nerve anastomosis may cause pain. Therefore, alternative safe and effective approaches are required. Previous studies have demonstrated that Schwann cell (SC) transplantation can accelerate neural regeneration and promote recovery of neural function but that implementation is difficult. 10 Sacrifice of functional nerves during SC isolation, problems associated with culture and expansion of SC populations in vitro, immunologic rejection, and proliferation and survival of SCs after transplantation into the injury site all decrease the feasibility of transplantation. 11,12 Recent studies have found that mesenchymal stem cells (MSCs) can promote neural regeneration and may be an ideal alternative. 13 Adipose-derived mesenchymal stem cells (ADSCs) have advantages compared with other kinds of MSCs that make them ideal seed cells for tissue regeneration and repair. ADSCs are easily harvested and are abundant in human adipose tissue. They continuously renew and are less immunogenic than other MSCs because they lack MHC class II or costimulatory molecules. 14,15 In animal studies and clinical trials, secretion of multiple nerve-growth factors following local transplantation of ADSCs promoted peripheral nerve repair and differentiation of neuronal cells. [16][17][18][19][20][21][22] The novelty of stem cell therapy and the need for regulatory approval have limited the available clinical applications, but Tonnard et al have described the preparation of nanofat, which is enriched in ADSCs, obtained by a purely mechanical procedure, and can be administered by injection with a 27 gauge needle. 23 Nanofat is an autologous product that does not contain any exogenous substances. Previous studies have described the successful use of nanofat for tissue repair and regeneration, anti-aging treatment, and the treatment of scars. 24,25 There are currently no reports of the application of nanofat for nerve regeneration after free perforating flap procedures.
Nerve growth factor (NGF) regulates the survival, growth, and differentiation of nerve cells in the peripheral and central nervous systems both during development and afterward. 26 In China, mouse (m)NGF has been used clinically to treat peripheral nerve injury, 27

| Preparation of nanofat
Nanofat was prepared by the Coleman technique as previously described and with the patient under anesthesia. 23

Lessons learned
• Nanofat plus mouse nerve growth factor (mNGF) injected into the deep dermis of anterolateral thigh (ALT) free flaps promoted sensory recovery.
• The mechanism may be associated with stimulation of angiogenesis and neurogenesis.
• Injection should be conducted when the flap has established a good-working microcirculation.
• The optimal mNGF solution/nanofat ratio was 1:9 and the optimal injection volume was 0.5 mL/cm 2 .

Significance statement
Nanofat and mouse nerve growth factor (mNGF) had a syn-

| Sensory evaluation before and after treatment
Before, 2, and 10 months after treatment, sensory function was evaluated by static two-point discrimination (2PD) and Semmes-Weinstein F I G U R E 1 Indicated injection areas on surface of a flap. The flaps were marked with 1 cm squares and injection areas were marked by using red symbols. The flaps were given a slow injection of 0.5 mL/cm 2 suspension or saline into the deep dermis F I G U R E 2 Sensory evaluation using a static two-point discrimination (2PD) test and the Semmes-Weinstein monofilament (SWM) test. Patients with nanofat plus mNGF had the smallest 2PD distance (A) and SWM (B) compared with nanofat or mNGF alone or control patients 2 and 10 months after treatment, which show that recovery of sensory function was better in patients treated with nanofat plus mNGF than in the other three groups. Differences in the results obtained with nanofat and with mNGF alone were not significant. **P < .05  cal University, China. ADSC isolation and culture was performed as previously described. 15 Briefly, human adipose tissue was minced and F I G U R E 3 Angiogenesis in flaps was evaluated by immunohistochemical staining of CD31 at 2 months after treatment. There were no significant differences in the number of blood vessels in the four groups before treatment. More new blood vessels were seen in the flaps of patients treated with nanofat plus mNGF, nanofat alone, and mNGF alone than in controls. Angiogenesis was the most evident in flaps treated with nanofat plus mNGF. Differences in angiogenesis seen in flaps treated with nanofat and mNGF alone were not significantly different. **P < .05

| Multiline differentiation of ADSCs
In vitro differentiation was performed as previously described. 28 Adipogenesis and osteogenesis were assayed on day 21 by 1% oil red O and alizarin red S staining. Chondrogenesis was assayed on day 28 by 1% alizarin blue staining.

| ADSC proliferation assay
The effect of mNGF on ADSCs proliferation was assayed in passage-3 cells seeded at a density of 4 × 10 3 cells/well in 96-well culture plates.
The cells were cultured in DMEM supplemented with 10% FBS and 1% penicillin-streptomycin containing with 0.9, 0.09, or 0.009 μg/mL mNGF for 24 or 48 hours. The proliferation of cultured ADSCs was assayed with a Cell Counting Kit-8 (Sigma). The absorbance of culture media was measured at 450 nm using a multilabel counter (n = 3).
F I G U R E 4 Neuroregenesis in flaps was evaluated by immunohistochemical staining of S100 at 2 months after treatment. Few S100 + cells were seen in any flaps before treatment. More Schwann cells were seen in flaps treated with nanofat plus mNGF, nanofat alone, and mNGF alone than in controls. More Schwann cells were seen in flaps treated with nanofat plus mNGF than in those treated with nanofat alone and mNGF alone. **P < .05 2.9 | Enzyme-linked immunosorbent assay (ELISA) ADSCs were cultured in medium containing with 0.9, 0.09, or 0.009 μg/mL mNGF at 37 C in 5% CO 2. After 24 hours, the medium was replaced with serum-free medium and without mNGF. After a further 24 hours, the supernatants were collected and passed through a 40 μm syringe filter to remove cell and tissue debris. Nerve growth factor (NGF) and neurotrophin-3 (NT-3) in the supernatant were assayed using a Quantikine ELISA kit (Sigma-Aldrich) following the manufacturer's instructions.

| Recovery of sensory function
All flaps survived well without any complications and were followed-up for 10 months after surgery. As shown in Figure

| Nanofat plus mNGF increased neoangiogenesis in flaps
Angiogenesis was evaluated in flaps by immunohistochemical assay of CD31 expression in blood vessel endothelial cells at 2 months after surgery ( Figure 3). There were no significant differences in the number of blood vessels in the four study groups before surgery. More blood vessels were seen in flaps treated with nanofat and/or mNGF than in those treated with sterile saline. Flaps treated with nanofat plus mNGF had more new blood vessels than those treated with nanofat or mNGF alone.

| Nanofat plus mNGF increased neurogenesis in flaps
Neurogenesis in flaps was evaluated by immunohistochemical staining of S100 in Schwann cells at 2 months after surgery (Figure 4). Few S100 + cells were seen in the four study groups before treatment, but more Schwann cells were seen in flaps treated with nanofat and/or mNGF than in controls and more were seen in flaps treated with nanofat plus mNGF than in those treated with nanofat or mNGF alone.

| mNGF promoted ADSC proliferation and nerve growth-factor secretion
The effects of mNGF on ADSC proliferation were assayed by flow cytometry and multilineage differentiation. ADSCs were >90% positive for CD105, CD73, and CD90 and negative for CD34, CD11b, CD19, CD45, and HLA-DR ( Figure 5A). Positive Oil red O, alizarin red S and alizarin blue staining confirmed that ADSCs had differentiated into osteocytes, adipocytes, and chondrocytes ( Figure 5B). The dosedependent effects of mNGF on ADSC proliferation are shown in Figure 6. mNGF stimulated paracrine secretion of NGF by ADSCs responsible for neurogenesis in flaps. NGF levels were significantly higher in mNGF-treated ADSCs than in controls and increased with the mNGF dose ( Figure 7A). Secretion of NT-3 by ADSCs also increased with increasing mNGF concentration. NT-3 levels were significantly increased by mNGF at both 0.9 and 0.09 μg/mL compared with controls ( Figure 7B). Among them, NGF has been proved not only to promote peripheral nerve development and regeneration, and also to promote angiogenesis. 33,34 Moreover, mesenchymal stem cells in accompany with NGF have been proved to have a synergistic effect -on promoting peripheral nerve repair. 35 In vitro, our studies show that mNGF has positive effects on the proliferation of ADSCs and secretion more neurotrophic factors, which suggested a potential therapeutic applicatio with the synergism of the two.

| DISCUSSION
Nanofat is a liquid fat emulsion prepared by mechanical processing and was first described by Tonnard et al in 2013. 23 Adipocytes do not survive nanofat preparation, but nanofat does contain many ADSCs, which may be responsible for neovascularization in the recipient area following intradermal injection, and the promotion of angiogenesis that is important for peripheral nerve growth. 23,26,36 As F I G U R E 7 Enzyme-linked immunosorbent assay (ELISA) of nerve-growth factor (NGF) and NT-3 expression induced in adipose-derived mesenchymal stem cells (ADSCs) by increasing concentrations of mNGF. A, NGF expression was significantly higher in mNGF-treated ADSCs than in controls and increased in a dose-dependent manner. B, NT-3 expression was significantly higher with 0.9 and 0.09 μg/mL mNGF compared with control cells. **P < .05, ***P < .01, # P > .05 The effects of mNGF on adipose-derived mesenchymal stem cells proliferation. mNGF increased cell viability in a dose-dependent manner. **P < .05 a stem cell therapy, the advantages of nanofat include simple, rapid preparation with no need of isolation and cultivation in the laboratory.
Proliferation, differentiation potential, and stem cell properties are preserved in cell culture. 23 38 In the study, the patients' BMI were between 18.5 and 23.9 kg/m 2 , which were in the normal range. Therefore, we think it would not affect the efficacy of nanofat.
The standard injection protocol used in this study, the mNGF and nanofat volume ratio and the injection volume/cm 2 , was determined in a preliminary study. To determine the optimal injection volume, 0.3, 0.5, and 0.8 mL/cm 2 nanofat were evaluated. At 0.8 mL/cm 2 , the flap became pale because of reduced blood flow. At 0.3 and 0.5 mL/cm 2 the flap color remained red and without signs of an insufficient blood supply.
An injection volume of 0.5 mL/cm 2 was chosen because sensory recovery at 2 months was better than that seen following injection of 0.3 mL/cm 2 . To determine the optimal mNGF solution/nanofat ratio, This is the first report of the use of mNGF plus nanofat to promote the sensory recovery of the free flaps. Clinical assessment was by 2PD and SWM tests. Histological assessment of recovery was by immunohistochemical staining of CD31 and S100 before and after the free-lap procedure. Although the study was limited by a small number of cases and short follow-up, the results did demonstrate the benefits of combined treatment with nanofat and mNGF. Evaluation of additional patients with longer follow-up is ongoing. Moreover, more data is needed before injection of mNGF and nanofat becomes universally performed.

| CONCLUSION
In this preliminary study, we investigated the sensory recovery-

CONFLICT OF INTEREST
The authors declared no potential conflicts of interest.

DATA AVAILABILITY STATEMENT
The data used to support the findings of this study are included within the article.