Fat Graft with Allograft Adipose Matrix and Magnesium Hydroxide-Incorporated PLGA Microspheres for Effective Soft Tissue Reconstruction

Abstract

BACKGROUND:

Autologous fat grafting is one of the most common procedures used in plastic surgery to correct soft tissue deficiency or depression deformity. However, its clinical outcomes are often suboptimal, and lack of metabolic and architectural support at recipient sites affect fat survival leading to complications such as cyst formation, calcification. Extracellular matrix-based scaffolds, such as allograft adipose matrix (AAM) and poly(lactic-co-glycolic) acid (PLGA), have shown exceptional clinical promise as regenerative scaffolds. Magnesium hydroxide (MH), an alkaline ceramic, has attracted attention as a potential additive to improve biocompatibility. We attempted to combine fat graft with regenerative scaffolds and analyzed the changes and viability of injected fat graft in relation to the effects of injectable natural, and synthetic (PLGA/MH microsphere) biomaterials.

METHODS:

In vitro cell cytotoxicity, angiogenesis of the scaffolds, and wound healing were evaluated using human dermal fibroblast cells. Subcutaneous soft-tissue integration of harvested fat tissue was investigated in vivo in nude mouse with random fat transfer protocol Fat integrity and angiogenesis were identified by qRT-PCR and immunohistochemistry.

RESULTS:

In vitro cell cytotoxicity was not observed both in AAM and PLGA/MH with human dermal fibroblast. PLGA/MH and AAM showed excellent wound healing effect. In vivo, the AAM and PLGA/MH retained volume compared to that in the only fat group. And the PLGA/MH showed the highest angiogenesis and anti-inflammation.

CONCLUSION:

In this study, a comparison of the volume retention effect and angiogenic ability between autologous fat grafting, injectable natural, and synthetic biomaterials will provide a reasonable basis for fat grafting.

Introduction

Autologous fat grafting is a remarkable procedure supplementing soft tissue deficiency since it has lots of advantages, including less invasiveness, easy accessibility, and less donor site morbidity [1]. However, their clinical outcomes are frequently suboptimal, unpredictable, and sometimes produce unpleasant complications, such as oil cyst, fat necrosis, and calcification which were mainly presumed by vascular insufficiency of recipient site and/or large volume bolus fat graft [2, 3]. The new cosmetic techniques such as a micro-fat graft (tiny volume fat graft) technique have been introduced because large volume bolus fat graft, for regenerating soft tissue defects, has frequently resulted in complications, such as calcification and oil cysts. Furthermore, recent researches have focused to improve the retention of transplanted fat with a combination of various additives such as platelet rich plasma (PRP), stromal vascular fraction (SVF) [2, 3], hyperbaric oxygen (HBO) [4], and tissue engineering scaffold [5].

Among the new approaches for effective soft tissue reconstruction, allograft adipose matrix (AAM) has been recently investigated as a natural scaffold to promote adipose tissue regeneration [6]. AAM is obtained through decellularization of allogeneic adipose tissue to retain the structural and biochemical properties of native matrix that would give support of adipogenesis [7–10]. In recent studies, AAM would promote soft tissue reconstruction by preventing the ischemic necrosis inside grafted tissue [8–10]. Giatsidis et al. [8] investigated that the combination of external volume expansion method and AAM for soft tissue reconstruction results in high volume retention and tissue preservation by adipogenesis and angiogenesi.

Additionally, the various synthetic polymers have been applied as synthetic scaffolds, including poly (L-lactic acid) (PLLA), polyglycolic acid (PGA), poly (lactic-co-glycolic acid) (PLGA), polyethylene glycol (PEG), polyethylene terephthalate, polytetrafluoroethylene, and polyethylene [11–15]. Among them, PLGA was approved by the Food and Drug Administration (FDA) in the fields of medical devices [16]. The PLGA microsphere has been widely used for various tissue regeneration including soft tissue because of its great degradability, biocompatibility, and usability to control shape and size [14]. For a polyester-based synthetic biodegradable polymers formulation to be successfully translated clinically, it must be non-toxic after in vivo implantation. However, synthetic polyester polymers generate acidic byproducts while being hydrolyzed at implanting site in vivo. The acidic degradation products of polymer can cause inflammation and necrosis of the surrounding tissue [17–19]. To address this issue, magnesium hydroxide [Mg(OH)₂; MH] was adapted in this study. In our group’s previous studies, MH nanoparticles showed a great pH neutralization effect on acidic decomposition products from polyester polymers in various biomedical fields [13, 16, 18, 20, 21].

Therefore, in this study, for effective fat graft with natural extracellular matrix-based scaffolds, namely AAM, and synthetic biodegradable polymer scaffold (PLGA) were used as regenerative scaffolds [4, 5]. We attempted to combine fat graft with regenerative scaffolds to reduce the amount of fat graft for achieving a target volume, and at the same time to increase retention volume of fat graft. We compared the volume retention rate of each scaffold with fat graft, including characterization of AAM, PLGA, and PLGA/MH through in vitro degradation test, cell cytotoxicity, and wound healing. In addition, we investigated how these regenerative scaffolds affect fat graft survival and volume retention through an animal study.

Materials and methods

Materials

Poly(D,L-lactide-co-glycolide) (PLGA, lactide:glycolide = 50:50, Mw; 110 kDa) was purchased from Evonik Ind. (Essen, Germany). Magnesium hydroxide [MH, Mg(OH)₂] and polyvinyl alcohol (PVA, 87–90% hydrolyzed, average Mw; 30–70 kDa) were purchased from Sigma-Aldrich (St. Louis, MI, USA). Dichloromethane (DCM) was obtained from Daejung Chemicals (Siheung, Korea). Allograft adipose matrix (AAM) was processed by CG Bio Co. (Seongnam, Korea).

Fabrication of the PLGA microspheres

A water-in-oil-in-water (w/o/w) double emulsion-solvent evaporation method was employed. A solution of PVA (2 w/v% in deionized water) was prepared for the external aqueous phase, and 1 ml DCM containing 20 mg of PLGA was used as internal oil phase. The mixture was homogenized at 800 rpm for 1 min (Silverson, L5M-A) and dried for 150 min to completely evaporate DCM at room temperature. The prepared emulsion was sifted out using sieve to targeted sizes. Microspheres were then washed in deionized water for 4–5 times being centrifuged at 3500 rpm for 15 min removing impurities. Finally, the purified microspheres were put in liquid nitrogen then immediately lyophilized (− 80 °C) for at least 2 days.

The MH-loaded PLGA microspheres were prepared by dispersing MH (30 wt.%) in PLGA solution sonicating at an amplitude of 40% power for 30 s. Making PLGA/MH solution in advance is required to be injected into external aqueous phase. PLGA/MH microspheres were also fabricated following the above process. The MH encapsulation efficiency was evaluated using following formula:

$$MHencapsulationefficiency\left(\%\right)=\frac{\left(ActualmassofMHinmicrosphere\right)}{FeedmassofMH}\times 100]$$

Scaffold characterization and in vitro degradation test

The prepared microspheres were visualized by optical microscopy (CKX53, Olympus, Tokyo, Japan), and the size was determined with supplied software. The surface morphology of the scaffolds was observed using scanning electron microscopy (SEM; GENESIS-1000, Emcraft, Gwangju, Korea). The thermal property of the scaffolds was analyzed by a thermal gravimetric analyzer (TGA 4000, PerkinElmer, Waltham, MA, USA).

To assess the neutralization capacity of the PLGA/MH microsphere, the mass and pH changes were measured in 0.5 mL phosphate buffered saline (PBS) solution (pH 7.4) at 37 °C for 60 days. The change in the pH value of the PBS solution was evaluated using a digital pH-meter (FP20, Mettler-Toledo GmbH, Schwerzenbach, Switzerland). The residual weight was calculated after complete drying of the microspheres for 3 days under vacuum condition from the following equation [22]:

$$Residualweight\left(\%\right)=100-\left[\frac{\left(initialweight-finalweight\right)}{initialweight}\times 100\right]$$

Cell cytotoxicity assay

Human dermal fibroblasts (hDFs) were grown in Dulbecco's Modified Eagle's Medium (DMEM; Hyclone, Logan, UT, USA) containing 10% fetal bovine serum (FBS; Hyclone) and 1% antibiotic–antimycotic solution (Gibco, Grand Island, NY, USA) at 37 °C in a humidified atmosphere with 5% carbon dioxide (CO₂). The viability of the cells was determined using a live-dead viability/cytotoxicity kit (Invitrogen, Thermo Scientific Inc., Waltham, MA, USA) and the fluorescence images were obtained using LSM880 (Zeiss, Jena, Germany). D-Plus™ cell counting kit 8 (CCK-8) cell viability assay kit was obtained from Dongin LS (Seoul, Korea) following manufacturer’s instructions.

In vitro wound healing assay

Human dermal fibroblasts (hDFs) were seeded at a density of 2 × 10⁵ cells/well in 6 cell tissue culture plate and cultured to form a confluent monolayer. The layer of cells was scraped with 1 mL micropipette tip straightly to create an artificial wound. The culture plate was washed three times with PBS solution to remove the detached cell debris and then treated with the scaffolds using trans-well inserts in DMEM containing 1% FBS and 1% antibiotic–antimycotic solution for 24 h. Wound closure was calculated as a percentage of the initial wound area, quantitated using NIH Image J software.

Quantitative real-time PCR (qRT-PCR)

The gene expressions of an anti-inflammation and angiogenesis in vitro were analyzed by qRT-PCR. Total cellular RNA from hDFs was isolated using the AccuPrep^® Universal RNA Extraction Kit (Bioneer, Daejeon, Korea) following the manufacturer’s instructions. The cDNA form isolated RNA was synthesized using PrimeScript RT Reagent Kit (Perfect Real Time, Takara, Tokyo, Japan). The qRT-PCR was performed using each primer and SYBR Green PCR Master Mix (Applied Biosystems, Thermo Scientific Inc.). The expression of angiogenic and inflammation related genes was calculated with the 18S rRNA as a reference gene using the 2^−∆∆Ct method. The primers used were as follows: 18S rRNA: forward, 5’-gcaattattccccatgaacg-3’ and reverse, 5’-gggacttaatcaacgcaagc-3’; IL-6: forward, 5’-gatgagtacaaaagtcctgatcca-3’ and reverse, 5’-ctgcagccactggttctgt-3’; IL-1β: forward, 5’-tacctgtcctgcgtgttgaa-3’ and reverse, 5’-tctttgggtaatttttgggatct-3’; TNF-α: forward, 5’- agcccatgttgtagcaaacc-3’ and reverse, 5’-tctcagctccacgccatt-3’; MMP2: forward, 5’-caccaccgaggattatgacc-3’ and reverse, 5’- cacccacagtggacatagca-3’; VEGF: forward, 5’-actggaccctggctttactg-3’ and reverse, 5’-tctgctccccttctgtcgt-3’; αSMA: forward, 5’-ctgttccagccatccttcat-3’ and reverse, 5’-tcatgatgctgttgtaggtggt-3’; ANGPT2: forward, 5’-tttgtgctgagtctggttgc -3’ and reverse, 5’-tcgtgtacctggggtcgt-3’.

The gene expressions of adipocyte and angiogenesis in vivo were assessed by qRT-PCR same as in vitro evaluation. The primers used were as follows: PPARG: forward, 5’-gacaggaaagacaacagacaaatc-3’ and reverse, 5’-ggggtgatgtgtttgaacttg-3’; AP2: forward, 5’-cctttaaaaatactgagatttccttca-3’ and reverse, 5’-ggacacccccatctaaggtt-3’; VEGF: forward, 5’-caggctgctgtaacgatgaa-3’ and reverse, 5’-gctttggtgaggtttgatcc-3’; αSMA: forward, 5’-taacccttcagcgttcagc-3’ and reverse, 5’-acatagctggagcagcgtct-3’.

Fat preparation

The study was performed after approval by the CHA University Bundang CHA Medical Center Internal Review Board (CHAMC 2020-03-013-003). Lipoaspirate (approximately 50 cc) was obtained by manual liposuction (Colemann technique) in the patient who underwent abdominoplasty and processed in a sterile fashion. The lipoaspirate was centrifuged at approximately 1800 g for 3 min to separate the adipose tissue from the oil part and the stromal vascular fraction: oil component was discarded to obtain the processed adipose tissue.

Allograft adipose matrix preparation

A Subcutaneous adipose tissue was obtained from donated human adipose tissue of woman who underwent abdominoplasty. Allograft adipose matrix (AAM) was processed as follows. The tissue was placed in isopropanol (Junsei, Japan) and treated using a shaking incubator at 37 °C for 12 h twice. The tissue was washed three times with PBS solution for 30 min each. This was followed by treatment with 1% SDS solution (Sigma-Aldrich) for 4 h at room temperature and washed overnight while replacing the PBS solution several times. After treatment in 0.1% peracetic acid solution (Sigma-Aldrich) for 2 h, it was washed three times for 30 min with distilled water. Samples were lyophilized and pulverized with a laboratory blender (Waring Products Co., New York, NY, USA) to obtained ECM powder. The powder and distilled water were mixed in a 1:4 ratio and radiation sterilized [7, 23].

Animal study

The animal experimental protocols for the use of animals were approved by the Institutional Animal Care and Use Committee of CHA University (IACUC approved number 200074) and carried out under the guidelines of the IACUC. A total of eight male 8-week-old BALB/c nude mice (Orient Bio Co., Seongnam, Korea) were used for this study and allowed to acclimatize for 2 weeks before the experiments. We allocated fat graft and regenerative scaffolds into four groups, including fat graft alone (group 1), fat graft with AAM (group 2), fat graft with PLGA/MH (group 3), and AAM alone (group 4). Processed lipoaspirate and/or rehydrated scaffolds (total 0.4 ml injection/each site) were injected into the subcutaneous layer of equally allocated four sites on each dorsum of 8 athymic nude mice under inhalation anesthesia using 2% isoflurane (Terel solution, Hana Pharm, Seungnam, Korea) gas. The grafted materials were harvested after 8 weeks, and each specimen volume was evaluated.

Histological analysis

Grafts from BALB/c nude mice were harvested 8 weeks after the experiment. Grafted tissue samples were fixed in 4% paraformaldehyde for a minimum of 48 h. The samples were processed using the standard method and prepared for paraffin tissue slides. The paraffin sections of 5 µm thickness were stained with hematoxylin and eosin (H&E) and immunohistochemistry (IHC) using anti-perilipin-1 antibody (Abcam, Cambridge, UK, ab3526, 1:200 dilution) and anti-CD31 antibody (Abcam, ab28364, 1:200 dilution). The secondary antibody (Alexa 555 Invitrogen, A21428) was incubated at room temperature for 2 h and nuclear staining was performed using DAPI. Fluorescent signals from graft tissue were visualized using a Nikon Microscopy (ECLIPSE 50i, Nikon Inc., Tokyo, Japan) and slide scanner (Zeiss Axio scan). Images were acquired using an Olympus DP71 digital fluorescence microscopy.

Statistical analysis

All experiments were repeated at least three times. The results are shown as the means ± standard error of the mean (SEM). Statistically significant differences were evaluated by one-way ANOVA using GraphPad Prism 7.0 software (GraphPad Software, Inc., La Jolla CA, USA). p values < 0.05 were regarded as statistically significant.

Results and discussion

Scaffold fabrication and characterization

Degradable polymer microspheres have been widely used in the fields of medical devices and medical applications. Among them, polymeric microspheres have been utilized for numerous biomedical applications such as tissue engineering and regeneration [24], drug or cell delivery [14], and cancer therapy [25]. When the polymeric microspheres are used as a biomaterial safely in clinic, the particle size of the microspheres should be consistently controlled. If the particle size is too small (less than 40 μm), the microspheres can cause capillary embolism [26].

As shown in Fig. 1, the surface of the biodegradable scaffolds was analyzed through the scanning electron microscopy (SEM). Based on representative SEM images, both of microspheres were observed with uniform size and smooth surface. And the AAM was full of the fibers of extracellular matrix. The amount of MH encapsulated in the PLGA microspheres was analyzed using thermal gravimetric analyzer (TGA). The content of MH in PLGA/MH microspheres was 21wt.-%. Therefore, the initial weight loss of the PLGA/MH was shown faster than the PLGA group. To assess an acid neutralization of MH enclosed PLGA microspheres, pH value was evaluated during degradation for 60 days (Fig. 1D). In day 1, pH value slightly increased in the PLGA/MH microspheres group due to initial release of MH in the surface. However, during polymer degradation, it performed great neutralization against acidic byproducts, lactic acid and glycolic acid.

Fig. 1.

Scaffold characterization. A representative scanning electron microscopy (SEM) images of the PLGA microsphere, PLGA/MH microsphere, and AAM (scale bar = 50 µm). B thermal gravimetric analysis (TGA) thermograms of each scaffold. Change of C pH and D mass during in vitro degradation in PBS solution at 37 °C for 60 days

In vitro biocompatibility and anti-inflammatory effect of the biodegradable scaffold

To investigate cytotoxicity of the scaffolds in vitro, in Fig. 2A, calcein AM and ethidium homodimer 1 (EthD-1) stainings were conducted with hDF at 24 h. Because of its well-known biocompatibility of the PLGA and AAM, the EthD-1 positive cells indicating dead cells were observed rarely in all the microspheres even the PLGA only group. However, the morphology of hDFs was changed abnormally. In Fig. 2B, the cell viability was quantified by CCK-8 with same time point. In the only PLGA microspheres treated group, the cell viability significantly decreased than control due to its acidic degradation products (p < 0.01).

Fig. 2.

Biocompatibility of the scaffolds. A live-dead assay images at 24 h (scale bar = 500 µm). B cell viability of the hDFs onto each scaffold at 24 h in vitro. The differences were considered significant when NS = not significant (p ≥ 0.05), *p < 0.05, and **p < 0.01 (n ≥ 3)

Additionally, inflammation has been always considered as a critical challenge to develop the effective biomaterials. Quantitative real-time PCR (qRT-PCR) was conducted to determine the expression of pro-inflammatory cytokine genes by the biodegradable scaffolds using hDFs (Fig. 3). The effect of the scaffolds was assessed with indirect cell culture system using trans-well. As a result, the PLGA microspheres increased the expression of pro-inflammatory cytokine genes, interleukin-6 (IL-6) and interleukin-1β (IL-1β) compared to normal hDFs as a control group. However, the PLGA/MH microspheres restricted or approximated the expression levels of IL-6, IL-1β, and tumor necrosis factor-α (TNF-α) compared to control. Especially, the expression of IL-1β significantly increased in the PLGA microspheres compared to control (p < 0.001) and statistically significant decreased in the PLGA/MH (p < 0.05). There were no statistical differences of the pro-inflammatory gene expressions in the AAM group compared to control. Consequently, AAM was not significantly related to the inflammatory response, and microspheres fabricated only with PLGA induced inflammation, but PLGA microspheres containing MH can greatly attenuate inflammatory response.

Fig. 3.

Anti-inflammatory effects on the scaffolds using hDFs. Gene expressions of hDFs related to inflammatory mediator levels: IL-6, IL-1β, and TNF-α. The differences were considered significant NS = not significant (p ≥ 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n ≥ 3)

Wound healing and angiogenic effect of the biodegradable scaffold

To increase transplanted adipose tissue survival, various studies have been attempted to develop new grafting systems with sufficient angiogenic activity [27, 28]. We also hypothesized that these new vessels would grow into grafted site during scaffolds degradation to assist retention and prevent apoptosis of the grafted fat. As shown in Fig. 4, the biological ability of the scaffolds was investigated in aspects of angiogenesis and wound healing for grafted fat survival. Based on wound healing analysis, the wound closure rates also highly increased from 31.56 (in the control) to 53.98% in the PLGA/MH microspheres, and slightly increased to 39.14% in the AAM compared to control. The qRT-PCR was assessed to determine the expression of angiogenesis related genes on the scaffolds with hDFs. The expression levels of matrix metalloproteinase-2 (MMP2) slightly increased in the PLGA/MH and the AAM groups, and contrastively, significantly decreased in the PLGA microspheres. Vascular endothelial growth factor (VEGF) is known to be mainly involved blood vessel formation. The VEGF expressions notably increased in the PLGA/MH (p < 0.0001) and the AAM (p < 0.001). Interestingly, in the PLGA/MH group, all the angiogenic related mRNA expressions were upregulated; MMP2, VEGF, alpha smooth muscle actin (αSMA), and angiopoietin-2 (ANGPT2). Therefore, on the same side of Figs. 2 and 3, the PLGA only microspheres would be insufficient candidate as an implanting scaffold for fat graft.

Fig. 4.

Angiogenic effects on the scaffolds using hDFs. A, B Observation of wound healing effect using optical microscopic image and quantification (scale bar = 500 µm). C Gene expressions of hDFs onto the scaffolds related to angiogenesis: MMP2, VEGF, αSMA, and ANGPT2. The differences were considered significant when NS = not significant (p ≥ 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n ≥ 3)

Volume retention effect of the biodegradable scaffolds for fat grafting survival

To investigate the actual volume retention ability, we conducted fat implantation with biodegradable scaffolds into the dorsum of mouse. Figure 5A shows the schematic illustration of in vivo implantation. After 8 weeks grafting, the only fat group was mostly absorbed. In the groups II and III, implant retention rate was similar and the highest implant retention rate displayed in the group IV (AAM only group). In gross examination, specimens of groups I and II looked like fat tissues and those of group III exhibited a whitish yellow fat tissue. Watery contents were observed in the implanting site of group II (fat with AAM). On the other hand, the specimens of group IV demonstrated whitish mass surrounded vessels without identified capsule formation (Fig. 5B).

Fig. 5.

A Schematic illustration of in vivo implantation, B the representative gross appearance of the biodegradable scaffolds, and C residual volume for 8 weeks after grafting. *p < 0.05 and ***p < 0.001 (n ≥ 3)

The average percentage of retention volume is 12.5% in the only fat group, 37.5% in the fat with AAM group, 40% in the fat with the PLGA/MH microsphere group, and 50% in the only AAM group. Especially, the AAM and the microsphere group showed high volume retention compared to the only fat grafted group (Fig. 5C).

In vivo evaluations of the biodegradable scaffolds for fat grafting survival

In order to analyze the residual tissue, histological analysis was conducted by H&E and IHC stains. Based on H&E images, lipid droplet-like structure was observed in Groups I, II, and III (the fat with AAM or PLGA/MH groups). On the other hand, the AAM only group was full of fibers same as Fig. 6A and lipid droplet was not observed. In addition, perilipin-positive cells were distributed throughout the inside of the tissue in the group II (fat with PLGA/MH group). And a very small proportion of perilipin-positive cells were observed in the group III (fat with AAM group) because of its high-density fiber. To explore the effects of the scaffolds on neovascularization into grafted tissue, cluster of differentiation 31 (CD31) immunostaining was evaluated. The expression of CD31 (indicated with star mark*) was significantly elevated in the group II (fat with PLGA/MH) grafted tissue.

Fig. 6.

In vivo evaluation for adipogenesis and angiogenesis of the biodegradable scaffolds. A Histological analysis using H&E (scale bar = 200 µm) and immunofluorescence stain for perilipin (red, scale bar = 200 µm) and CD31 (red, scale bar = 50 µm). B–D Gene expressions related to adipocyte: PPARG, AP2; related to angiogenesis: MMP2, VEGF, αSMA, and ANGPT2; and proinflammation cytokine: IL-6, IL-1β, and TNF-α. The differences were considered significant when NS = not significant (p ≥ 0.05), *p < 0.05, **p < 0.01, ***p < 0.001, and ****p < 0.0001 (n ≥ 3)

In parallel, mRNA expression levels of adipocyte related genes peroxisome proliferator activated receptor gamma (PPARG) and adipocyte protein 2 (AP2) demonstrated higher in the group II (fat with PLGA/MH) (Fig. 6B–D) than the fat with AAM and the AAM only. Besides, we observed the upregulated expression of the angiogenic gene expressions, VEGF and αSMA in the group II. The pro-inflammatory cytokines (IL-6, IL-1β, and TNF-α) were also statistically downregulated in the group II (fat with PLGA/MH group). Overall, on the basis of in vivo evaluations, although the AAM has great volume retention ability, the PLGA/MH microspheres have not only capability of volume retention, but also anti-inflammation effect and angiogenic ability. These prominent advantages could support to increase fat grafting rate in clinic.

Conclusions

Fat grafting is one of most popular techniques to supplement the volume of soft tissue. For example, aging face, small soft tissue defects can be well corrected by fat grafting. Small volume fat grafts in the well vascularized area are well survived, but large volume fat grafts or fat grafts in the poor vascularized area have frequently shown oil cysts, calcifications within the grafted fat tissue.

Recently, AAM filler was developed and used for soft tissue augmentation. Several studies have demonstrated fat regeneration within implanted AAM materials [6, 29, 30]. It sounded like a fancy material replacing fat grafting. In the contrary of those results, our study, unfortunately showed that fat regeneration was rarely found in the groups of fat graft combined with AAM and AAM only implantation. AAM showed outstanding ability to maintain the implanted tissue volume as a filler to augment the soft tissue, but lack of capability to regenerate the fat tissue.

Besides AAM, in the group of fat graft combined with the PLGA/MH, increased volume retention was demonstrated compared to the fat only group, and the PLGA/MH microsphere further increased their vascularity to assist long-term survival (volume retention) of autologous adipose tissue grafts in a murine model.

This study accessed the role of additive materials (PLGA/MH microspheres and AAM) in fat grafting. The PLGA/MH and AAM increased volume retention in comparison with only fat grafted group, and the PLGA/MH microspheres further attenuated inflammation and enhanced their vascularity to assist volume retention of autologous adipose tissue grafts in a murine model for 8 weeks. It could prevent to heading toward apoptosis of the grafted tissue. This study has limitation that the result showed a little bit short outcomes to evaluate the regeneration of the fat tissue. Therefore, our further study will be explored for extremely long-term evaluation of PLGA/MH microspheres in vivo with the retention volume, vascularization, and apoptosis on the grafted tissue. Taken together, this study demonstrates that the PLGA/MH microspheres can be a great additive biomaterial for fat graft.

This study demonstrates that additive scaffolds have a potential to increase the volume retention of fat graft with increase of vascularity or volume augmentation effect. Therefore, for the soft tissue defects required large volume supplement, fat graft with additive biocompatible scaffold such as the AAM or PLGA/MH seems to be more effective and less complicated than large volume fat graft.

Declarations

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical statement

The study was performed after approval by the CHA University Bundang CHA Medical Center Internal Review Board (CHAMC 2020-03-013-003) and carried out following the guidelines for animal experimentation of the Animal Institutional Review Board of CHA University CHA Bundang Medical Center (IACUC 200074).