Use of concentrate growth factors gel or membrane in chronic wound healing: Description of 18 cases

Abstract

Treating chronic skin wounds in patients with diabetes, bed sores, or stasis dermatitis is typically a time‐consuming and costly process, and the outcome is not always promising. Concentrated growth factor (CGF) obtained from the autologous venous blood of patients via fractional centrifugation is employed for producing a CGF gel or membrane that can be applied to expedite self‐regeneration of skin wounds. In this case report, we presented the results from 18 patients with chronic skin wounds treated with a CGF gel or membrane produced from autologous venous blood. Noticeable granulation tissue and regenerated epidermal coverage were observed in 16 patients who received CGF treatment over various time courses, thereby demonstrating the significant therapeutic effects of CGF treatment in overall wound healing. The other two patients with stasis ulcers in their calves failed to respond to the treatment because of the comorbidity of iliac vein thrombosis. In addition, by culturing HaCaT keratinocytes using CGF membrane as the foundation, we observed that HaCaT cells attached to the CGF membrane migrated and proliferated to form an epithelium‐like structure. Comprehensively, the clinical results infer that CGF gel can expedite the regeneration of the soft tissue at the wound, whereas CGF membrane may facilitate its marginal re‐epithelialisation. The combination of the two can promote autologous regeneration of both deep and superficial wounds effectively and safely.

1. INTRODUCTION

Platelet‐rich plasma (PRP) is widely applied in autologous regenerative medicine as a growth pool of growth factors and cytokine.1 Different PRP preparations elicited different responses.2 Concentrated growth factors (CGF) gel may be considered as an upgraded version of PRP and was prepared by drawing venous blood in sterile vacuum tubes without anticoagulant solutions.3 These tubes were immediately centrifuged in the Medifuge MF200 machine (Silfradent S.R.L., Forli, Italy). CGF gel is composed of a fibrin network, which contains activated platelets, leukocytes (including CD34 positive cells), and growth factors (such as TGF‐β and VEGF).3, 4 Advances in regenerative medicine have uncovered a distinct potential of CGF gel in wound healing5, 6 and dentistry.7, 8 It has also been suggested that interfollicular injection of autologous CD34 positive cell‐containing PRP preparation has a positive therapeutic effect on male and female pattern hair loss without remarkable major side effects.9 Preparation of CGF gel does not require any anticoagulants, thrombin, or calcium chloride. In regenerative medicine, CGF gel may be applied as a multifactorial stimulation system.

We now present a case report of the application of autologous CGF gel or membranes to promote healing in chronic wounds with only epidermal loss or even full thickness soft tissue loss (Figure 1).

Figure 1.

Deep ulcer in dorsum of right hand of a 76‐year‐old female (case 12) before (A), during (B), and after (C) CGF treatment

2. CASE REPORT

One male and five female patients with diabetic ulcers, five male patients with shallow stasis erosions, one male and two female patients with deep stasis ulcers, one female and two male patients with pressure sore, and one female patient with full thickness soft tissue defect after cellulitis were included in this report (Table 1). The maximal width, length, and depth of wounds were measured by Vernier calliper (Sellery Inc., Arcadia, California). Sixteen patients were treated and recorded until the chronic wounds showed complete reconstruction. Two patients were treated and recorded until the chronic wounds showed no significant reconstruction after undergoing the CGF therapy at least five times. The details of all patients and treatment courses are listed in Table 1.

Table 1.

Clinical characteristics of patients

Pt.	Sex/age	Site of lesions	History of wound/mo	No. of CGF gel treatment	No. of CGF membrane treatment	Maximal width × length/mm	Maximal depth/mm	Duration of treatment/wk	Scar formation	Combined disease
1	Male/72	Right leg	4	0	1	42.3 × 35.6	1.3	2	No	Stasis erosion
2	Male/92	Right buttock	12	0	1	11 × 11	1.3	3	No	Pressure sore
3	Male/67	Right leg	1.5	0	2	11.1 × 1.4	1.4	4	Yes	Stasis erosion
4	Female/64	Right leg	1	0	1	18.2 × 16.5	1.5	3	No	Diabetes
5	Female/87	Scaral skin	18	0	1	62.2 × 25.6	1.5	3	No	Pressure sore
6	Male/49	Left ankle	1	0	1	38.5 × 32.1	1.5	2	No	Stasis erosion
7	Male/65	Right leg	3	0	2	42.5 × 37.5	1.5	4	No	Stasis erosion
8	Male/68	Right leg	2	0	2	58.5 × 55.5	1.5	4	No	Stasis erosion
9	Male/74	Left knee	3	0	2	31.5 × 12	2	6	No	Diabetes
10	Female/70	Right second toe	3	0	3	12 × 12	2.5	10	No	Diabetes
11	Female/66	Right leg	1	0	3	37.3 × 6.2	2.5	8	Yes	Diabetes
12	Female/58	Left leg	1	3	2	20.5 × 30.5	3.2	14	Yes	Stasis ulcer
13	Female/70	Left leg	1	3	3	17.5 × 16.3	3.5	16	No	Diabetes
14	Female/76	Dorsum of right hand	1	2	1	32.5 × 26.6	4	6	Yes	Cellulitis
15	Female/57	Right heel	8	4	2	30 × 25	4.3	32	Yes	Diabetes, uraemia
16	Male/80	Sacrum	6	4	2	16 × 9	16	14	Yes	Pressure sore
17	Female/67	Left leg	10	2	5	24 × 19.5	3.3	18	Failure	Stasis ulcer,left iliac vein thrombosis
18	Male/69	Right leg	96	5	0	40 × 25	4	13	Failure	Stasis ulcer,right iliac vein thrombosis

Abbreviation: CGF, concentrated growth factor.

In this study, 14‐28 mL of whole blood was phlebotomised from each patient. The volume of blood drawn depended on the area and depth of wounds in each patient. The application of CGF gel or membrane in patients was performed as described by Rodella et al.3 The CGF gel was produced as follows: 7 mL of blood was injected into each sterile serum blood collection tube (BD Vacutainer, REF 367820, Becton Dickinson and Company, Franklin Lakes, New Jersey). These tubes were immediately centrifuged in special machine (Medifuge CGF MF200100 Silfradent S.R.L., Sofia, FC, Italy). After centrifugation, three blood fractions were obtained: (a) the upper platelet poor plasma layer; (b) the middle fibrin‐rich gel with aggregated platelets and CGF gel layer; and (c) the lower red blood cells layer (Figure 2).

Figure 2.

Deep pressure sore in mid sacrum of a 80‐year‐old male (case 13) before (A, B, C), during (D), and after (E) CGF treatment

The CGF gel layer was separated and transferred into a petri dish under a laminar flow hood. The CGF gel was compressed by pliers to create a sheet of CGF membrane. The application of CGF gel or membrane for wound treatment was first described by Romano et al.4 We modified the procedures of treatment according to the different stages of wound healing. For superficial chronic erosions, only the CGF membrane treatment was used. For deep chronic ulcers, the CGF gel treatment was initially used, but was later discontinued on the appearance of overgrowing granuloma in lesion. Instead, CGF membrane treatment was used to cover the granulomatous tissue until complete re‐epithelialisation was achieved. At the same time, we used liquid nitrogen cryospray (5 seconds over 1 cm² skin area, once a week) to inhibit the overgrowing granulomatous tissue until complete re‐epithelialisation of the wounds was achieved.

Before performing the CGF treatment, bacterial infections of all chronic wounds should be brought under control. Initially, the wounds were cleaned and debrided. Then CGF gels or membranes were inserted into the cavity of ulcers or onto the erosions and were covered with an occlusive dressing (Duoderm Extra Thin CGF Dressing. Convatec Inc., Greensboro, North Carolina). The occlusive dressing was changed every 3‐4 days (Figure 3).

Figure 3.

Deep diabetic ulcer in right leg of a 66‐year‐old female (case 11) before (A), during (B) and after (C) CGF treatment

The procedure of CGF gel or membrane wound treatment as described above was repeated for each per 2 weeks and continued until completely reconstructed wound healing. The preventive antibiotic was prescribed during treatment (Anicyn, 625 mg/Tab, 1^#, bid, China Chemical & Pharmaceutical Co, Ltd.), and discontinued when there were no signs of infection or inflammation.

In the treatment of nine patients with chronic erosive wounds or shallow chronic ulcers (1‐2 mm in depth), only 1‐2 applications of CGF membrane were required to achieve satisfactory cosmetic appearance of re‐epithelialisation in chronic wounds (2‐6 weeks of treatment) (Table 1).

In the treatment of seven patients with chronic ulcerative wounds (2‐16 mm in depth), even with full thickness soft tissue defects, it took 3‐12 applications of CGF gel or membrane treatment to achieve satisfactory cosmetic results (6‐32 weeks of treatment in seven patients). The delay in wound healing was more significant in patients with diabetic or stasis ulcers (Table 1). In the treatment of two patients with deep stasis ulcers and extensive stasis petechiae, no significant improvement was observed even after 5‐7 applications of CGF treatment. The two patients were also diagnosed with either right or left iliac deep vein thrombosis (Table 1).

During the chronic wound treatment, overgrowth of granulomatous tissue and scar formation was observed in five cases (Table 1). We applied liquid nitrogen spray to inhibit the overgrowth of granulation tissue and to prevent scar formation. Then we covered the above wounds with the CGF membrane to promote re‐epithelialisation.

These cases showed that the time required for chronic wounds to heal with CGF treatment corresponds to (a) the wound depth instead of the wound area or (b) the existence of combined diseases such as diabetes or chronic venous insufficiency (Table 1). In the treatment of impaired wound healing, the CGF therapeutic model has proven to be an efficient and safe autologous multifactorial stimulation system with minor scar formation.

Using CGF membrane as the foundation of cell culture for HaCaT cells (Figure 4).

Figure 4.

CGF gel (A) is produced by using the techniques of autologous venous blood sampling and fractional centrifugation. CGF gel is pressed by a clipper to produce CGF membrane (B). Before transplanting, the structural image of tri‐dimensional network of CGF membrane composed of fibrin under inverted microscope (C). HaCaT cell suspension (1 × 10⁴ cells/mL in DMEM) is transplanted onto the surface of the CGF membrane and is completely covered by the cell suspension (D). After transplanting HaCaT cells to the surface of the CGF membrane, they are co‐cultured for 14 days. Then, CGF membrane with attached and proliferated HaCaT cells can be obtained (E, F). The section of CGF membrane co‐cultured with HaCaT cells showed fibrin clot with an epithelium‐like tissue is formed by multiple layers of HaCaT cells being stacked over the roof of the CGF membrane and a single layer of HaCaT cells at the bottom of CGF membrane (G). When CGF membrane and HaCaT cells are co‐cultured in vitro, CGF membrane can act as the foundation for HaCaT cell proliferation and movement. It is proposed that autologous CGF membrane can promote marginal re‐epithelialisation in the healing of chronic wounds (H). CGF, concentrated growth factor; DMEM, Dulbecco's modified eagle medium

HaCaT cells provided by the Department of Dermatology of Kaohsiung Medical University were cultured on a CGF membrane. The CGF membrane was constructed using the blood taken from the same healthy adult male (Figure 4A,B). Initially, cell suspension made from HaCaT cells was added to the CGF membrane so as to cover the whole membrane (Figure 4C,D). After letting the dish sit still for 8 hours, the entire petri dish (35 mm) was filled with a medium such that the air‐fluid surface did not exceed the top surface of the CGF membrane. The same culturing process was repeated three times and samples were separately collected. The medium used in the culture was Dulbecco's Modified Eagle Medium/Low Glucose (Hyclone, SH30021.01), 10% fetal bovine serum (Hyclone, SH30088.03), and penicillin 100 IU/mL as well as streptomycin 100 μg/mL (Hyclone, SH30010). The cell culture was maintained at 37°C, 5% CO₂, and the culture medium was changed every 3 days. After culturing the cells for 14 days, the CGF membrane that the HaCaT cells had grown and attached upon (Figure 4E,F) was removed for tissue sectioning and haematoxylin and eosin staining. It can be observed that epithelium‐like tissue is formed by multiple layers of HaCaT cells being stacked on the roof of the fibrin clot of CGF membrane, and a single layer of HaCaT cells at the bottom of CGF membrane (Figure 4G).

3. DISCUSSION

The process of normal wound healing requires the coordination of many factors, for example, activated platelets, neutrophil, monocytes, and macrophages along with a moderate amount of growth factors, cytokines, and chemokines.10, 11 In chronic ulcerative wounds such as diabetic ulcers, stasis ulcers, and pressure sores, the presence of an abnormal part or atypical wound healing processes may be the reason behind poor wound healing.10

As per this report, when CGF gel or membrane is used to cure chronic wounds, three major clinical phenomena can be observed in the following order:

1. New granulation tissue with red spots will grow from the bottom of the wound and the new granulation tissue will gradually fill the wound and could even exceed the height of the peripheral normal skin.

2. The regeneration of epithelial tissue begins from the periphery of the wound that is adjacent to normal skin and grows toward the centre to finally cover the entire wound.

3. In cases of successfully treated chronic ulcers using CGF gel or CGF membrane, only hypertrophic or atrophic scars are observed, and there is no keloid formation.

In addition, when CGF membrane is used as the foundation for cell culture with HaCaT cells added on the top of it, an epithelium‐like tissue will form by multiple layers of HaCaT cells getting stacked over the roof of the fibrin clot of CGF membrane after approximately 2 weeks.

By summarising the results of the above‐mentioned clinical case treatment and in vitro coculture of HaCaT cells with CGF membrane, we propose the possible mechanisms for CGF gel or membrane in treating chronic ulcers as follows:

1. Possible mechanisms that promote the generation of granulation tissue:

   1. In the process of producing CGF gel, fractional centrifugation can cause platelets to release their growth factors and cytokines, which can facilitate wound healing.12, 13, 14, 15
   2. The stem cells and monocytes present in the CGF gel can move to the bottom of the wound, proliferate, and differentiate further into macrophages to facilitate wound healing.16, 17, 18

2. The epithelialisation process is impaired in all types of chronic wounds.19 The development of CGF membrane may promote re‐epithelialisation and wound closure facilitated by the 3D fibrinoid structure of CGF membrane20 and the growth factors it contains.21, 22 It is proposed that CGF membrane may act not only as a biological barrier23 but also as a source of growth factors and a foundation for epithelial cells to attach, migrate, and proliferate (Figure 4H). These can promote re‐epithelialisation in superficial or deep chronic wound healing.

Platelet‐based autologous therapies have created a new field in regenerative dermatology.24, 25, 26, 27 There are many products to choose from, when using platelet‐based autologous therapies. Several factors need to be considered:

1. If the subject using the therapy has any comorbidities;

2. The objective of using the therapy, that is, the type of disease to be treated, and if PRP is to be used alone or if other blood components will also need to be isolated and used;

3. The amount of PRP aggregates needed for one‐time usage, that is, the amount of blood that needs to be processed;

4. If the subject undergoing the therapy or local ordinance permits the addition of non‐autologous agents (eg, CaCl₂ or thrombin) into the autologous blood to be used for regeneration treatment;

5. The expense of a single treatment and if the patient can afford it;

6. If the manoeuvre of the therapy is simple enough and the time‐span needed for a full course of treatment.

Compared with other platelet‐based autologous therapies, the benefits of using CGF gel or membrane to cure chronic wounds are as follows:

1. The entire process of producing CGF gel is rapid and simple: Only one fractional centrifugation for approximately 15 minutes is required to complete the production of the CGF gel. Regardless of the size and depth of the wound, only enough venous blood drawn from the patient to cover the wound is required. The whole process can be completed in 60 minutes.

2. The entire CGF treatment is non‐invasive and safe.

   1. Withdrawing autologous venous blood from the patient to produce CGF gel can prevent possible infection or rejection from heterologous transplant.
   2. Addition of chemicals (eg, CaCl₂) is not required to stimulate platelet activation. The activation of platelets is caused by the physical process of fractional centrifugation.
   3. Anaesthesia is not required during autologous transplantation. It is ideal for patients who are not suitable for general anaesthesia surgery (eg, patients with acute myocardial infarction who need flap graft).

3. The successful rate in treating chronic wounds is high and the occurrence of hypertrophic scars is reduced.

4. The CGF autologous treatment model provides a more economical way to treat chronic wounds; its manoeuvre is simple, safe, effective, and can reduce scar formation. The material fee of CGF gel or membrane treatment is approximately US$70‐100, depending on the size and depth of the wound. It is much cheaper than the medical expenses charged for autologous flap graft surgery.

5. Patients with a variety of chronic wounds such as stasis ulcers, diabetic ulcers, and pressure sores can choose CGF for treatment on the premise that: (a) The arterial blood supply and venous blood return surrounding the chronic wound are as normal as possible; it would not be suitable if there is serious venous or arterial thrombus in the periphery of the wound. (b) Necrotic tissue in the chronic wound must be fully debrided before implementing CGF gel transplant to let the CGF gel have direct contact with the live tissues.

The selection of treatment method for chronic wounds is summarised as follows: (Figure 5)

1. For treating superficial chronic wounds (Figure 5AE), using CGF membrane to cover the wound (Figure 5BE) is suggested until complete re‐epithelialisation of the epithelium is achieved (Figure 5CE).

2. For treating deep chronic wounds (Figure 5A), a two‐stage treatment is suggested. The first stage begins following complete debridement of the wound (Figure 5B).Sufficient autologous CGF gel is used to fill the wound (Figure 5C) and the wound is tightly covered with occlusive dressing. This procedure is repeated until regenerated granulation tissue fills the entire wound (Figure 5D). The second stage begins when the deep wound is filled with regenerated granulation tissue and its height is slightly more than the surface of the surrounding skin. At this time, the CGF gel grafting is stopped and liquid nitrogen spray is used to inhibit further growth of the regenerated granulation tissue (Figure 5E). CGF membrane is then used to cover the wound (Figure 5G) until re‐epithelialisation of the whole epithelium is completed (Figure 5H).

Figure 5.

Schematic drawing of the usage of CGF gel or CGF membrane to cure chronic wounds is shown. For deep chronic ulcer wounds (A), the necrotic scar tissue must first be debrided until some bleeding spots appear (B). The autologous CGF gel is transplanted onto the wound (C). Platelets in the CGF gel will release various growth factors and cytokines to promote regeneration of granulation tissue (D). When the regenerated granulation tissue gradually fills the whole wound and its height exceeds the surface of the surrounding normal skin, liquid nitrogen spray is used to inhibit its further growth (E), and the CGF gel grafting into the wound is no longer required and is replaced by CGF membrane covering (F) so as to promote marginal re‐epithelialisation and facilitate wound healing (G). Finally, the regeneration and migration of epithelial cells completely covers the wound (H). The regenerative repair of dermis will continue until wound healing is complete (I). For superficial chronic erosion wounds, it is suggested that the wound be debrided (AE) and directly covered with CGF membrane (BE) to promote marginal re‐epithelialisation of the wound (CE) until the entire epithelium tissue is completely regenerated and healed (I). CGF, concentrated growth factor

It is anticipated that in the future, CGF gel or membrane would be used as a three‐dimensional scaffold for autologous in‐vitro culture in combination with adipose‐derived stem cells and CGFs (such as PDGFs, bFGF, VEGF, IGF‐1, and TGF‐β) released by PRP collected from autologous blood samples and thereby promote its application in the different fields of autologous regenerative medicine.28

Kao C‐H. Use of concentrate growth factors gel or membrane in chronic wound healing: Description of 18 cases. Int Wound J. 2020;17:158–166. 10.1111/iwj.13250