April 2020 HSC Section 4 - Plastic and Reconstructive Problems

The Journal of Craniofacial Surgery

Volume 30, Number 3, May 2019

Brooker et al

Given the ASC and stromal stem-cell population, it was theo- rized that SVF enrichment of adipose tissue grafts may improve graft volume retention. A number of animal studies, some of which are summarized in Table 1, compared graft retention rates of adipose/SVF grafts to adipose grafts alone and overwhelmingly reported superior rates in the adipose/SVF cohorts. Matsumoto et al published one of the earliest studies using an immunodeficient mouse recipient model for human adipose grafts. 32 It was found that the addition of SVF significantly improved graft retention rates and through immunofluorescent tagging of the SVF cells, they were able to later identify them between mature ASC in the connective tissue and some expressed Von Willebrand factor, suggesting increased angiogenesis was one part of their mechanism of action. 32 Another study by Paik et al used a similar model but employed varying doses of cells ranging from 0 (control) to 10 million and found that graft retention rates at higher doses of SVF were poorer which the authors hypothesized was due to increased metabolic demand from the increased loading of stem cells which compete for nutrients with the adipocytes in the postgraft period. 33 Rasmussen et al included 13 animal studies for SVF-enriched adipose grafting in their review and found a 0.65- to 2.5-fold improvement in volume retention compared to adipose grafting alone after a mean of 13 weeks. 34 Human studies have been reported using SVF-enriched grafts to the face. In their review, Toyserkani et al included 3 studies of SVF- enriched facial fat grafting, summarized in Table 2, all of which led to significant increases in volume retention compared to fat grafting alone. 35 In their meta-analysis, Zhou et al included 4 studies using SVF-enriched fat grafting to the face and found a significant overall increase in volume retention in the SVF-enriched group (71%) compared to the unenriched group (52%). 36 They also found that the number of reoperations was reduced from 24.2% to 10.6%. 36 Gontijo-de-Amorim et al published a prospective comparative study of 30 patients with hemifacial soft tissue volume loss from tumor excision, Parry-Romberg syndrome, and trauma. 37 Fifteen patients received standard autologous fat grafting while the remain- ing 15 received grafting enriched with SVF. At 12 months a subset of 5 patients from each group underwent volumetric analysis by computed tomography (CT) and all had aesthetic outcomes judged by independent surgeons at 1 to 2 years. 37 Example patient images (both with Parry-Romberg syndrome) prior to grafting and 2 years after grafting are shown in Figure 3, with the top patient receiving only un-enriched grafting and the bottom patient receiving SVF- enriched grafting. Patients receiving SVF-enriched grafting were judged to have an ‘‘excellent’’ outcome by the independent surgeon panel in 82.5% of patients while those receiving graft only were judged ‘‘excellent’’ in 47.6% of patients. The CT volumetric analysis showed a volume loss of 24% in the un-enriched control group and 9.6% in the enriched group. Flow cytometry identified an average of 16,000 mesenchymal stem cells in each of 25 SVF pellets analyzed. 37 Facial rejuvenation has also been examined using SVF-enriched grafts. Charles de Sa´ et al harvested abdominal fat from 6 facelift patients and extracted SVF from the lipoaspirate. 38 Patients received SVF-enriched fat to the right preauricular area while expanded ASC in culture (number of passages unknown) was administered to the left preauricular area. Skin biopsies were assessed 3 months after grafting using histologic and electron microscopy analysis. 38 Important considerations for the use of ASC enriched fat grafts are the regulatory constraints, especially in the United States and European Union, and the added cost of the procedure versus fat grafting alone. Unlike mechanical emulsification, deliberate sepa- ration of SVF is considered ‘‘greater than minimal’’ manipulation in many regions and the cell product has a higher regulatory burden.

Cultured ASC-Enriched Fat Grafting The ASCs represent a more homogenous population of cells compared to SVF after culturing. 48,49 A number of animal studies specifically examined ASC enrichment have been reported. For example, Zhang et al harvested adipose from donor rats and extracted the SVF before culturing in fetal bovine serum. 42 The ASCs were isolated and expanded to passage 4, mixed with adipose grafts and injected into recipient rats. CD34 staining was greater at 14 days in the ASC-enriched grafts and volume retention was greater at 3 months than nonenriched grafts. 50 Piccinno et al utilized a rabbit model and harvested SVF from animals from which they then isolated ASC via 4 sequential passages. 43 These ASCs were mixed with hyaluronic acid and autologous adipose grafts and implanted. The ASC-enriched grafts showed greater vascularity and reduced necrosis at 14 days and after 3 months; ASC-enriched grafts also showed significantly greater volume retention. 43 Mose- ley et al published one of the few studies in mice comparing both cultured ASC and fresh ASC-enriched grafts in mice. Both were reported to be superior in volumetric retention and qualitative appearance (abundance of adipocytes and lower fibrosis than con- trols) and the transgenic ASCs were still present upon immunohis- tochemical staining at 6 months. 44 Rasmussen et al reviewed 11 animal studies using cultured ASC with 8 reporting significant improvement in volume retention over adipose grafting alone. 34 In general, the studies reported greater vascularity and decreased necrosis cysts on histology in the ASC-enriched grafts. 34 Human studies with ex vivo expanded ASC are less numerous than those for SVF-enriched adipose grafts; however, a number do exist. Kølle et al performed the first randomized controlled trial whereby patients underwent 2 separate liposuction procedures, 1 to harvest adipose for injection, and the other for isolation of ASCs. 54 The 2nd aspiration was split into 2, with 20 million ASCs added to 1 half only. The ASC-enriched and un-enriched grafts were then randomly injected into the posterior part of the right and left upper arm. Volume was measured by MRI at the time of engraftment and 121 days later where the ASC-enriched graft was found to have a retention of 80.9% versus 16.3% for the un-enriched graft. 54 Koh et al applied this to the face in 5 subjects with Parry- Romberg syndrome, with 5 further subjects receiving microfat injections only as controls. 55 Donor fat was obtained from abdomi- nal lipoaspirates and cultured for 14 days whereupon microfat in the experimental group was enriched with 1 10 7 cells. Patients under- went CT volumetric analysis at 6 months which showed a 2.51-fold fat uptake rate in the experimental versus control group. Figure 4 shows the front-on views taken preoperatively, 6 months postoper- atively, and 12 postoperatively showing long-term retention though no control group images were included by Koh et al. At present at least 1 human trail examining the utility of ex vivo expanded ASC in facial fat grafting is currently underway with no results posted at the time of writing (NCT03258164). Barriers to clinical application of this technology include not only the time and resource intensive process of ex vivo expansion of primary stem cells but also regulatory hurdles as well. Currently, human cell based therapies are allowed to be marketed only with FDA approval unless they meet certain stringent parameters including minimal manipulation, something which all ex vivo expanded cultures have undergone. 64 At present a significant amount of rigorous evidence from controlled trials will be required before this technology can be effectively marketed. More radical utilization of ASC has been reported in the repair of osseous craniofacial defects. Multiple in vivo studies in rats, rabbits, and mice have been reported some of which are summarized in Table 1. These studies have shown inconsistent results, with some showing considerable efficacy using implantable matrices seeded

# 2019 Mutaz B. Habal, MD

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