Adipose-derived Stem Cells Added to Platelet-rich Plasma for Chronic Skin Ulcer Therapy

Introduction. Adipose-derived stem cells (ASCs) hold great promise for regenerative medicine applications due to their ability to promote the healing process through in situ differentiation and secretion of paracrine factor. The aim of this paper is to present a clinical adjunct for chronic skin wound therapy based on ASCs added to platelet-rich plasma (PRP), to obtain an enhanced PRP (e-PRP).Materials and Methods. For 18 months, 24 control-group patients with 31 chronic skin ulcers were treated with standard wound care, while 16 experimental-group patients with 21 chronic skin ulcers were treated with standard wound care and 1 e-PRP injection. The patients were randomly assigned to the control or experimental group. Outpatients had weekly follow-up visits where they were subjected to standard treatment and the wound healing process was assessed. Results. At the end of the study, the control and experimental groups had similar healing rates but wound closure rates were significantly different (P = 0.0257): 0.0890 cm² x day and 0.2287 cm² x day respectively, resulting in a faster recovery for the group treated with e-PRP. No side effects were reported. Conclusion. In the authors’ experience, e-PRP significantly enhanced wound closure rates when compared to standard wound care, without causing any serious complications. This finding highlights e-PRP as a valuable resource for chronic wound treatment. 

A dipose-derived stem cells (ASCs) are particularly promising for regenerative therapies¹ as they can be harvested easily through standard liposuction procedures with minimal donor site morbidity.² These stem cells have a differentiation potential similar to other mesenchymal stem cells as well as a higher yield upon isolation and a greater proliferative rate in culture when compared to bone marrow-derived stem cells.^3-5 Moreover, as ASCs do not necessitate extensive manipulation before application, compliance with “cell manufacturing” in accordance with current Good Manufacturing Practice Guidelines is not required.⁶ In effect, these restrictions do not apply in European countries in cases of minimal manipulation [Regulation (EC) No 1394/2007 of the European Parliament and of the Council]. 

Platelet-rich plasma (PRP) is defined as platelet concentration above baseline normal platelet count in a small volume of plasma;⁷ it derives from whole fresh blood through double-spin centrifugation and has healing anti-inflammatory and proregenerative properties.^8,9 The aim of this study was to describe a combined application of autologous ASCs and PRP to obtain an enhanced-PRP (e-PRP) for the treatment of chronic skin ulcers

Surgical technique. During a same-day surgery procedure, ASC isolation and e-PRP injections were performed. On the morning of surgery, a 42-cc blood sample was taken from each patient in five 6-mL collection tubes (BD Vacutainer K2E tubes, Plymouth, UK) with anticoagulant citrate dextrose-A (ACD-A) and in two 6-mL serum collection tubes (Vacutainer CAT tubes, BD, Plymouth, UK). The blood in the ACD-A-containing tubes was fractionated using centrifugation (Heraeus Megafuge 1.0 ml; Thermo Scientific, Langenselbold, Germany) (210g for 10 minutes) to obtain the PRP. While the serum tubes were centrifuged (2560g for 10 minutes), the supernatant plasma was removed under a sterile hood, added to calcium gluconate (1:10), incubated for 30 minutes at 37°C, and centrifuged at 836g for 15 minutes to obtain the supernatant serum rich in thrombin. The platelets were concentrated in the PRP at approximately 10⁴/µL ± 20%, as it has been shown that a concentration of approximately 4-7 times more than the usual baseline platelet count produces clinical benefits, while higher concentrations do not appear to result in a greater effect.^9,10 The resulting PRP and platelet-poor plasma (10 cc) were stored at room temperature until needed (about 40 minutes).

A vibrating shaker (Multi Reax, Heidolph, Schwabach, Germany) and centrifuge (MPW 223, Johnson & Johnson Medical, New Brunswick, NJ) were both used in the operating room and placed in a laminar airflow bench (1200 FLO, Nuova FIMS srl, Concorezzo (MB), Italy) to isolate the ASC pellet as previously described.¹¹ In brief, the abdominal subcutaneous adipose tissue was harvested using a standard liposuction procedure by means of 400 mL of Klein solution as tumescent volumes, a 3.0 blunt-tipped cannula and 10-mL syringes (Luer-Lok syringe, BD, Franklin Lakes, NJ). Eighty mL of adipose tissue were usually harvested and subsequently positioned in the vibrating shaker at 600 vbr/min for 6 minutes and centrifuged at 52g for another 6 minutes. The ASCs obtained were poured into a 10-mL syringe containing 5 mL of previously collected autologous PRP: the e-PRP was ready to be injected into the skin edge as well as at the bottom of the lesion itself. 

The wound dressing was made with the remaining 5 mL of PRP. This was activated by adding autologous thrombin produced by mixing the autologous phosphoprotein phosphatase (PPP) with a calcium chloride solution (CaCl₂ 1 mL 10%; 1000 units: 1 mL; ratio PRP/PPP:10/1) to obtain the platelet gel that was inserted into the surgical field as needed, and kept until the fifth postoperative day. The total procedural time was 45 minutes. 

Patients were enrolled when informed consent was obtained from each study participant with chronic venous, diabetic, and ischemic ulcers. Exclusion criteria were patients on chemotherapeutic agents; patients with abnormal laboratory test levels including hemoglobin < 10.5 g/dL, platelet count < 100 x 10³/µL, or serum albumin level < 2.5 g/dL; wounds due to malignancy; and wounds with active clinically diagnosed infection.

To assess the effectiveness of the e-PRP, 40 patients were enrolled (mean age: 70 years; 21 males, 19 females) and randomly divided into 2 groups, both of which were treated with the same advanced dressings. Only the experimental group was treated once with e-PRP.

The control group included 24 patients (mean age: 74.5 years; 10 males and 14 females), who had 31 chronic skin ulcers, 42% of which were of arterial etiology, 45% consequent to venous insufficiency, 10% diabetes related, and 3% posttraumatic. Mean wound size was 11.24 cm² (SD = 2.6) at baseline. The chronic wounds included in the study had an average duration of 14.5 months (SD = 5.22).

The e-PRP group population included 16 patients (mean age: 70.75 years; 11 males and 5 females); there were 21 chronic skin ulcers, mean wound size was 25.18 cm² (SD = 5.6) at baseline. The chronic wounds included in this group had an average duration of  26.57 months (SD = 6.39). The wound was arterial in 67% of included cases, venous in 19% of the cases, and of diabetes-related etiology in 14% of the cases.

This 18-month study was conducted in compliance with the Declaration of Helsinki and the Guidelines for Good Clinical Practice. All enrolled patients provided written informed consent before inclusion in the study. All patients were subjected weekly to standard wound treatment, which consisted of disinfection, debridement when needed, and application of advanced wound dressing and bandages.

Pictures of the lesions were taken at the onset of the study (T₀), and then at 1, 3, 6, 12, and 18 months. Area measurements were performed on the digital photographs by means of digital planimetry using the VistaMetrix v1.33 (SkillCrest LLC, Tucson, AZ) software. First the wound sizes at T₀ were analyzed, then the wound closure rate, which was obtained by dividing the ulcer size at T₀ by the number of days needed for healing (cm²/day). 

Data are expressed as mean ± SD. Statistical comparisons between the groups were performed using Student t test. A value of P < 0.05 was considered statistically significant. 

Both the platelet count in the blood and in the PRP was studied, as well as the number of ASCs isolated on average for each procedure, to be able to correlate the results obtained with the effectiveness of the technique. 

At the end of the study, the chronic skin wounds of 68% (n = 21) of the control group and 71% (n = 15) of the e-PRP group healed completely (Figures 1, 2, 3 and 4).

Analysis between the 2 groups was performed on all data collected but no statistical significance was found; hence only the healed patients of both groups were assessed.

The mean wound onset values were 10.19 months (SD = 4.37) for the control group and 14.53 months (SD = 1.859) for the e-PRP group, distance between means was 4.343 ± 3.501, and no statistically significant differences emerged (P value = 0.2233). 

Mean wound sizes were: 8.519 cm² (SD = 2.902) for the control group and 29.59 cm² (SD = 9.752) for the e-PRP groups, distance between means was 0.1397 ± 0.05987; t test highlighted a statistically significant difference (P = 0.0236).

The wound closure rate of the control group was 0.0890 cm²/day (SD = 0.01871), while that of the e-PRP group was 0.2287 cm²/day (SD = 0.06617). The t test confirmed the difference was statistically significant (P = 0.0257).

After the procedure, some patients complained of discomfort in the abdominal region for a few days but no longer than 1 week, which is a common side effect of liposuction,¹² but no further side effects were reported. 

The mean baseline platelet count was 244 x 10³/µL while the platelet concentration in PRP ranged from 960 x 10³/µL to 135 x 10⁴/µL. Starting from 80 mL of adipose tissue, 5 x 10⁵ ASCs (5% of the total number of sample cells) were collected on average, with a cell survival rate of 97%; while the remaining 95% of cells were mostly blood-derived and endothelial cells. 

Adipose-derived stem cells have diverse clinical applications, including the treatment of nonhealing wounds,^13-18 while PRP is an advanced wound therapy that has been used in hard-to-heal acute and chronic wounds for more than 20 years.^8,19,20

The authors’ clinical experience with the use of e-PRP shows not only the effectiveness of the procedure but also its feasibility. In effect, it takes only 45 minutes  to perform, with minimal patient discomfort. Moreover, the safety of e-PRP is guaranteed by the use of only autologous components.¹¹

Platelet-rich plasma not only works as a vehicle for ASCs but may also act synergistically with stem cells through its growth factor, influencing both resident and newly brought cells.²¹ However, it must be specified that a major bias in this study was the impossibility of determining the exact, single contribution to the healing process of ASCs and PRP when injected individually. Further studies are needed to assess whether such a coadministration synergistically improves the effects of ASCs and PRP alone.

Chronic wounds and their treatment pose a great medical challenge for patients as well as for the health care system. Regenerative medicine and cell-based therapy recently have tried to respond to the need for new methods of enhancing the healing process to achieve optimal outcomes.²² In this field, ASCs are the stem cell of choice due to their abundance, the minimally-invasive harvesting procedure, high yield upon isolation, and easy clinical application with no prior need to be manipulated.^23,24 Since preliminary results of this e-PRP therapy for chronic wounds are promising, taking into account the therapy’s effectiveness and safety, the authors believe this might be a useful therapeutic approach for hard-to-heal wounds. 

From the Department of Surgical Sciences, Plastic Surgery Division, University of Parma, Italy; Cutaneous, Mininvasive, Regenerative and Plastic Surgery Unit, Parma University Hospital, Parma, Italy; Department of Clinical and Experimental Medicine, Division of Hematology, Parma University Hospital, Parma, Italy; and ⁴Diagnostic Department, Immunohematology and Transfusion Unit, Parma University Hospital, Parma, Italy 

Address correspondence to:
Edoardo Raposio, MD, PhD, FICS
Department of Surgical Sciences, Plastic Surgery Division and Cutaneous, Mininvasive, Regenerative and Plastic Surgery Unit,
Parma University Hospital
Via Gramsci 14, 43126,
Parma, Italy
edoardo.raposio@unipr.it or
edorap@yahoo.it

Disclosure: The authors disclose no financial or other conflicts of interest.