A Systematic Review of Autologous Platelet-Rich Plasma and Fat Graft Preparation Methods.

BACKGROUND: The addition of platelet-rich plasma (PRP) to adipose tissue may improve fat graft survival, although graft retention rates vary markedly between studies. To what extent this outcome heterogeneity reflects differing methodological factors remains unknown. This systematic review aims to synthesize and critically review methodological approaches to autologous PRP and fat cotransplantation in both human and animal studies.
METHODS: In accordance with PRISMA guidelines, Ovid MEDLINE, Scopus, and Cochrane Library databases were searched from inception to April 2017. Data were extracted from all in vivo studies involving autologous PRP and fat cotransplantation. A secondary aim was to assess reporting of technical detail; authors were not contacted to provide missing data.
RESULTS: From 335 articles, 23 studies were included in the qualitative synthesis. Some 21 were performed in humans and 2 in rabbits. Six studies were randomized control trials; the remainder reported on observational data. Methods of PRP extraction and activation varied markedly between studies. Fat graft preparation was comparatively more consistent. Methods of PRP and fat mixing differed significantly, especially with regards to relative volume/volume ratios.
CONCLUSIONS: Our study represents the first systematic review of methodological factors in autologous PRP and fat cotransplantation. It demonstrates that technical factors in graft preparation and administration vary significantly between in vivo studies. Such methodological heterogeneity may explain observed differences in experimental and clinical outcomes. Reporting of key procedural information is inconsistent and often inadequate. These issues make meaningful evaluation of the PRP-enhanced fat grafting literature difficult and may limit its translation into clinical practice.

INTRODUCTION

Autologous fat grafting is used extensively in reconstructive and aesthetic plastic surgery for the correction of soft-tissue volume defects. The popularity of structural free fat transfer comes from the fact that it is nonimmunogenic, versatile, and easily harvested with low donor-site morbidity.[1]

More recently, there has been growing interest in the regenerative potential of free fat transfer.[2,3] Multipotent precursor cells transplanted within the fat stromal compartment—termed adipocyte-derived stem cells (ASCs)—are able to differentiate into various cell lineages[4] and secreting soluble mediators with angiogenic, immunosuppressive, and antiinflammatory properties.[5] Several preliminary studies have demonstrated that autologous free fat transfer may significantly enhance healing of diabetic foot ulcers,[6] pressure sores,[7] and radiotherapy scars.[8]

However, long-term fat graft retention is highly variable with resorption rates in the order of 25% to 80%.[9-11] Fat necrosis and subsequent graft resorption is therefore a significant problem. Various technical factors related to fat preparation and administration have been implicated in graft failure.[12-15] Similarly, adequate neovasculascularization appears to be an equally important prognostic factor in graft survival. Adipocytes poorly tolerate ischemic conditions,[16] and early angiogenesis is essential for successful transplantation.[17-19]

Platelet-rich plasma (PRP) is an autologous source of concentrated platelets, growth factors, and cytokines used widely in regenerative medicine.[20-24] Its therapeutic efficacy derives from the supraphysiological release of several growth factors and bioactive proteins from platelet α granules.[17,25] These mediators—including platelet-derived growth factor, vascular endothelial growth factor, transforming growth factor β, and insulin-like growth factor—have 2 primary effects: they promote the recruitment and activation of cells responsible for tissue repair, and they encourage new blood vessel formation.[22]

Independently, both PRP and fat transplantation seem to have a role in regenerative medicine; however, the addition of PRP to fat transplants may synergistically enhance graft survival by a number of mechanisms. First, the secretion of proangiogenic factors by PRP may improve early graft vascularization and minimize ischemic injury[26,27]; second, the release of antiinflammatory cytokines is thought to have a role in preventing graft degeneration[27]; and third, PRP-secreted factors have been shown to enhance the differentiation of preadipocytes into their mature form.[28,29]

The majority of human and animal studies suggest that PRP and fat cotransplantation increase graft survival rates. However, the rate of graft resorption varies markedly between studies and, in particular, between research groups. Furthermore, several human[30-32] and animal[33] studies report that the addition of PRP to fat transplants confers no survival advantage when compared with free fat transfer alone. To what extent these variable outcomes reflect differing methodological factors remains unknown.

For PRP alone, different study methodologies produce context-dependent results.[34-36] PRP preparation techniques, composition, mechanism of activation, and outcome measurements vary significantly.[23] This inconsistency is further confounded by poor reporting of key technical factors.[24,37,38] Together, these issues limit the ability to draw overall conclusions about the efficacy of PRP in each setting and hinder its translation into clinical practice. Similarly, methodological heterogeneity in the harvesting and processing of fat transplants affects both experimental[39-42] and clinical[15] outcomes.

It is therefore reasonable to expect that methodological factors in PRP-enriched fat grafting will be important determinants of transplant survival rates. As in individual PRP and fat transfer studies, there is—as yet—no standardized transplantation protocol for their combined use.[24,43,44] Preliminary evidence from both in vitro[28,45] and animal studies[46,47] supports the hypothesis that technical factors significantly influence PRP–fat cotransplantation outcomes.

This systematic review aims to synthesize and critically review the various methodological approaches to autologous PRP and fat cotransplantation in both human and animal studies. In particular, it seeks to assess to what extent technical factors in PRP and fat processing vary between comparable studies.

METHODS

The objective of this systematic review is to critically evaluate the literature on PRP-enriched fat grafting in accordance with PRISMA guidelines.[48] It aims to provide a descriptive synthesis and critical appraisal of technical factors in autologous PRP and fat cotransplantation.

Search Methods

Bibliographic databases (Ovid MEDLINE, Scopus, and The Cochrane Library) were searched for relevant articles from inception to April 2017. Both “free-text terms” and “MeSH term” searches were run sequentially to capture all papers in which PRP was coadministered with fat. Variations on the following keywords were combined using the matrix of Boolean operators detailed in Supplemental Digital Content 1: “platelet-rich plasma,” “PRP,” “platelet concentrate(s),” “fat graft(s),” “fat transfer,” “fat injection(s),” “mixed,” “method(s),” “extraction,” “preparation,” “activated,” “human,” “animal,” “autologous.” English language studies from any country of origin were eligible for inclusion. (See appendix, Supplemental Digital Content 1, which displays the electronic database search matrix, http://links.lww.com/PRSGO/A622.)

After merging database search results and discarding duplicate entries, titles and abstracts were screened to eliminate studies unrelated to the current research objective. The remaining articles were read in full to assess their eligibility for qualitative synthesis. Additional articles were identified by routinely screening the reference lists of all included studies. All authors agreed upon the final list of included studies; any disagreement between authors was resolved by discussion and consensus.

Study Selection Criteria

A full set of inclusion and exclusion criteria were prespecified and agreed upon by all authors in advance. This study protocol was not eligible for registration on the PROSPERO database[49] as it constitutes a review of methodological factors only.

All in vivo studies in which autologous PRP was mixed or coadministered with autologous fat grafts were eligible for inclusion. Both preclinical and clinical trials performed for any surgical or medical indication were included. All methods of fat grafting (such as lipofilling, contouring, and solid tissue transplant) were deemed eligible. No differentiation was made between pure PRP and leucocyte-rich PRP.[50] All experimental study designs (irrespective of number of fat grafts or length of follow-up) were included.

In vitro studies only (without a concurrent in vivo human or animal model) were excluded. Studies in which PRP or fat grafting had been performed separately (ie, without mixing or coadministration) were excluded. Animal studies using a pooled isogenous source of PRP were ineligible for inclusion. Studies reporting solely on platelet-rich fibrin (even if added to fat grafts) were excluded. Similarly, those studies involving only secondarily extracted fat products (ie, the stromal vascular fraction or isolated ASCs) were discarded. Case reports, letters, editorials, book chapters, and reviews were also excluded.

During the shortlisting process, articles were screened for reporting of technical and methodological factors related to PRP and/or fat processing and coadministration. Articles with either a comprehensive or partial description of their methods were included; studies with no methods were excluded. One of the secondary aims of this study was to assess whether authors were reporting sufficient technical detail in their published manuscripts; therefore, no additional contact was made with authors to provide missing data.

Data Collection

All data were extracted by a single author (JL) and recorded on a predesigned bespoke electronic form before being independently verified by a second author (OJS).

Data collected were divided into the following subgroups:

baseline study data (title, author(s), year, journal, country, in vivo model)

experimental details (study design, number of fat graft procedures, length of follow-up)

PRP extraction (whole blood extraction methodology, PRP processing steps, PRP activation details, mean platelet counts, PRP storage)

fat extraction (method of fat harvest, donor-recipient site combination, fat processing methods, fat storage)

PRP–fat mixing (method and timing of PRP–fat mixing, use of prosurvival adjuncts).

No assumptions were made about the methods used if they were not explicitly reported. If authors referenced supplemental materials, appendices, or other articles in their methods, these were reviewed for relevant data extraction. Methodological factors not reported either directly or indirectly were listed as “unspecified.”

In studies with multiple different experimental protocols, data were extracted from those related to the current research objective only.

Quality Assessment and Statistical Analysis

All studies fulfilling the inclusion criteria were eligible for inclusion, and low-quality studies (eg, those performing poorly according to GRADE criteria[51]) were not discarded.

The purpose of this study was to provide a descriptive summary and critical review of variation in experimental technical factors—no attempt to account for risk of bias either in or between studies has been made.

Formal evaluation of experimental and clinical outcomes was beyond the scope of the current research objective; therefore, we have made no attempt to perform pooled statistical comparisons or meta-analyses. Owing to significant study design heterogeneity, such quantitative comparisons would be fundamentally misleading. Where appropriate, we have indicated which studies demonstrate improved graft retention rates with autologous PRP and fat cotransplantation and which do not.

RESULTS

Study Selection

The electronic database search strategy returned 335 results. After the elimination of 58 duplicates and the inclusion of 7 additional records, 286 titles and abstracts were screened against the predefined research objectives. At this stage, 203 articles were excluded; 83 papers were read in full to assess their eligibility for inclusion.

During this process, 60 manuscripts were eliminated for the following reasons: either PRP (n = 21) or fat (n = 10) was used in isolation; transplants were obtained from nonautologous sources (n = 8); reviews, letters, and abstracts (n = 15); no reporting of methods (n = 2); single case reports (n = 4); in vitro experimental studies only (n = 3), or the use of cultured ASCs only (n = 1).

In total, data were extracted from a final shortlist of 23 articles included in the qualitative synthesis. A comprehensive study attrition diagram is provided in Fig. 1.

Fig. 1.

Study attrition chart.

Study Characteristics

Of the 23 shortlisted studies, 21 were performed in humans and 2 in rabbits. The majority of human studies were presented as observational case series, although many included a comparator control arm of some description; only one[52] was explicitly described as a case-control study. Four human studies used a randomized control trial design, as did both animal experiments.

All included studies were published since 2009. Studies performed in 4 different continents were included; 11 included articles were published by 2 interrelated Italian research groups.[29,53-62]

Data were extracted from only those experimental protocols where PRP was combined with fat for cotransplantation. The number of grafts fulfilling the inclusion criteria and length of follow-up are provided in Table 1.

Table 1.

Characteristics of Included Studies

PRP Extraction

Methods of PRP extraction varied significantly, particularly between research groups (Table 2). The majority of human studies employed a commercially available PRP extraction system—in these 17 studies, 5 different manufacturer devices were used, each with their own protocol and PRP product characteristics.[35,36,63] Only 4 human trials (and both animal studies) did not use a commercial device to produce PRP.

Table 2.

PRP Extraction Methods

Most studies used a citrate-based anticoagulant to obtain whole blood; 9 studies did not report on their choice of anticoagulant. Where citrate was used, reporting on its strength and volume/volume (v/v) ratio to blood was inconsistent and often omitted (data not shown).

The volume of whole blood collected varied between studies and depended largely on the anticipated volume of PRP required. However, 16 studies did not specify what volume of PRP was produced for a given volume of blood; in the 9 studies where these data were reported, the ratio of whole blood to PRP varied from 2:1[31] to 13:1.[52] Overall, only 4 studies[52,64-66] provided comprehensive whole blood collection details.

Reporting of PRP centrifugation protocols was generally better, with only 5 studies providing inadequate methodological detail.[52,54,64,67,68] Most studies used a single centrifugation spin, although the relative centrifugal forces used and the length of time doing so varied between different study designs. Interestingly, this was not necessarily a consequence of using different commercial PRP extraction systems—for example, 4 studies using a commercial RegenKit device employed different centrifugation parameters.[31,54,56,69] Only 2 studies quantified mean PRP platelet concentrations[32,69] before fat grafting; none of the other studies provided either absolute PRP concentrations or change from baseline data.

PRP Activation

Most studies included a PRP activation step (Table 3)—2 human studies did not use activated PRP[52,70] and 2 studies did not report on whether or not activated PRP was used.[57,69]

Table 3.

PRP Activation Methods

Studies using commercial devices typically followed manufacturer instructions for activation using calcium chloride, autologous thrombin, or both. However, of the 17 studies using commercial PRP systems, only 2 provided methodological detail within their published manuscript.[54,66] Of the 5 studies using a bespoke extraction method in which activated PRP was used, 2 did not describe the steps involved.[67,71]

In general, authors added the activation reagent to PRP immediately before admixing with fat. Where PRP was injected into the area of fat grafting in vivo, activation was performed either immediately before percutaneous injection[66] or not at all.[52]

The majority of studies extracted PRP at the point of use and did not store PRP in either its active or inactive form for any significant length of time. Where PRP was stored for a short period during fat harvest, it appears that this occurred at room temperature—only one author described maintaining PRP in an ice water bath before implantation.[52]

One study with an optimized experimental PRP extraction protocol activated PRP with calcium chloride and thrombin before cryogenic storage at −80°C.[52] To complete the freeze-thaw cycle, the authors then recentrifuged the product at 18°C to achieve a platelet concentration of 1.4–1.9 × 106 platelets/μl.

Fat Preparation

Fat preparation methods were more uniform between studies (Table 4). All human studies used a version of the Coleman liposuction technique,[72] excluding one[70] in which fat was obtained using microsuperficial enhanced fluid fat injection. Abdominal fat harvest (either with or without additional donor sites) was common to all human studies excluding one[66] where only the thigh was used. In the rabbit animal study using liposuction harvest, adipose tissue was obtained from the groin fat pad.[64] With the exception of the other animal study—in which adipose tissue was harvested from the scapular region and grafted to the ear using an open surgical approach[71]—all studies transplanted fat and PRP via percutaneous injection.

Table 4.

Fat Preparation Methods

Several authors commented on a decantation step before fat centrifugation.[32,52,64,68] Centrifugation was typically performed for 3 minutes at 3000 rpm. Only 2 studies did not include a fat centrifugation step—in both cases, the authors used either saline[69] or Ringer’s lactate[70] to clean the tissue before transplantation. Only one study[52] described routine fat storage—in this case using an ice bath for between 2 and 4 hours to cool fat before implantation, although no rationale was provided for this decision.

Recipient sites varied according to the indication. Most study designs involved a single recipient site for fat grafting (eg, for gluteal augmentation[65] or facial lipostructure[32,52,67]); other study designs were based around an indication for fat grafting, irrespective of target site (eg, for scar improvement[56,68]); 2 studies compiled data from unrelated patient series.[59,60]

PRP–Fat Grafting

Most studies admixed PRP and fat in sterile syringes immediately before cotransplantation. Three studies[52,66,68] transplanted PRP and fat separately, injecting PRP into the fat compartment after lipofilling. One animal study[71] soaked each 0.8 g adipose tissue fragment in 1 ml of activated PRP (for an unspecified length of time) before transplantation.

Relative PRP and fat volumes varied significantly between studies (Table 5). Several studies used a 1:1 ratio of PRP to fat,[32,64,71] whereas others used as little as 0.1 ml of PRP per ml of fat.[31,65-67] Many study designs used a range of PRP volumes for a given quantity of fat (eg, between 0.2 and 0.5 ml of PRP per ml of fat[29,53,56,57,60-62]). Only 3 of these studies provided a rationale for this: 2 were attempting to identify the optimal in vivo v/v concentration[29,60] and 1 selected PRP volume according to the size of the recipient site.[61]

Table 5.

PRP and Fat Cotransplantation Methods

Three studies (2 in humans[52,69] and 1 in rabbits[64]) routinely used prophylactic antibiotics. Several studies used additional regenerative adjuncts for either some or all of their grafts, including nonablative laser therapy,[56,68] hyaluronic acid–medicated biologic dressings,[54] and delayed percutaneous graft site insulin injections.[29]

DISCUSSION

Our article represents the first systematic review of methodological factors in autologous PRP and fat cotransplantation. It demonstrates that technical factors in graft preparation and administration vary significantly between in vivo studies. It also highlights that reporting of key procedural information is inconsistent and, in many cases, inadequate.

Evidence from both the fat[12,14,73] and PRP[35,36,63] literature suggests that methodological factors are critical determinants of experimental and clinical outcomes. It is therefore reasonable to assume that these procedural elements will be equally important in PRP and fat cotransplantation. Most studies included in this systematic review found that the addition of PRP to fat improves graft retention rates[29,52-62,64-71]; however, 3 articles[30-32] report no such survival advantage. It is possible that this variability is at least partially explained by differing study methodologies.

The lack of consistent reporting of essential technical factors further compounds the interpretation of such outcome heterogeneity. Without a minimum description of the qualitative and quantitative characteristics of the PRP–fat transplant used, it is impossible to draw meaningful cross-comparisons between studies.[23] The absence of sufficient experimental methodological detail or standardized preparation protocol may limit the translation of PRP-enhanced fat grafting into clinical practice.

For PRP alone, various authors have proposed descriptive classification taxonomies based upon either preparation methods[23] or final product constituents.[50] The absence of an accepted methodology for the preparation and activation of PRP reflects ongoing disagreement in the preclinical literature.[44] Several experimental studies have attempted to optimize this process,[25,74] but consensus has yet to be reached. The growing use of commercial devices—each with their own unique PRP product characteristics[34,35,63,75]—further compounds the issue.

Comparatively, the preparation of adipose tissue for structural fat grafting is less controversial, and most included studies employed a version of the Coleman liposuction technique.[72] However, greater variability is seen in the v/v mixing ratio of PRP to fat, with up to 10-fold differences in substrate concentrations used. Again, this reflects a lack of consensus in the preclinical literature. Two studies found that a PRP volume fraction of 5% provided the optimal environment for ASC proliferation in vitro[28,45] (with paradoxical inhibition of ASC proliferation at higher concentrations[28]). However, these findings have not been replicated elsewhere: 1 study found that 20% PRP v/v promoted maximal graft viability,[47] whereas a different research group identified a dose-dependent effect for concentrations up to 50%.[29] This inconsistency likely reflects intertrial heterogeneity of the PRP product used.[47] It is also worth considering that, in most clinical scenarios, the most appropriate v/v concentration will be derived from the minimally effective ratio of PRP to fat. In the case of large volume fat transplantation, higher ratios (for example, like those approaching 1:1 in the 2 included animal studies[64,71]) would otherwise require the use of an unfeasibly large volume of whole blood.

This systematic review provides a comprehensive descriptive analysis of the current methodological approaches to in vivo autologous PRP and fat cotransplantation. It is based upon a robust, multiperson search strategy with additional article retrieval from reference lists, but it remains possible that potentially eligible studies were not identified. During the protocol stage, we designed a set of inclusion criteria to address our current research objective; studies using nonautologous and isogenous sources of PRP were excluded, as were trials using second-generation PRP products (such as platelet-rich fibrin). Similarly, experimental approaches using secondarily extracted fat components only (including the stromal vascular fraction and isolated ASCs) were deemed beyond the scope of this review. Finally, we have focused on critically reviewing methodological factors in PRP and fat cotransplantation but have not evaluated other areas that could influence experimental outcome (including outcome measure selection or indication for intervention).

CONCLUSIONS

Methodological factors in autologous PRP-enhanced fat cotransplantation vary significantly between in vivo studies. This is confounded by inconsistent and inadequate reporting of essential procedural information and combined PRP–fat transplant characteristics. The absence of sufficient methodological detail makes meaningful evaluation of the PRP-enhanced fat grafting literature difficult.

PRP and fat cotransplantation is an innovative and potentially transformative therapeutic strategy in regenerative medicine. However, a failure to address the fundamental issues identified in this review may limit its translation into clinical practice.