Effect of platelet rich plasma gel in a physiologically relevant platelet concentration on wounds in persons with spinal cord injury

Abstract

The objective of the study was to investigate the use of a 1·3 times normal platelet concentration platelet‐rich plasma (PRP) gel to move chronic wounds towards healing in persons with spinal cord injury (SCI). The study design was a case series of 20 persons with SCI with non healing wounds. The outcome measures were, in wound area, volume, undermining and sinus tracts/tunnels (ST/Ts), calculated average, (i) percent of change from baseline, (ii) change per day from baseline, (iii) number of treatments and (iv) number of weeks. In a mean of 4·0, after treatments over 3·4 weeks, the wounds closed on an average of 47·9% in area and 56·0% in volume. Undermining closed on an average of 31·4% using 3·5 treatments over 2·6 weeks. ST/Ts closed on an average of 26·1% after 2·3 treatments over 1·5 weeks. Clinical relevance by percent of positive responders and their response: in area, 90·0% of the subjects responded positively, the average reduction was 53·8%. In volume, 90·0% responded, with an average reduction of 67·3%. Of four subjects with undermining, 75% closed 47·0% on average. Of the three with ST/Ts, 100% closed 26·1% on average. Average haemoglobin and haematocrit levels were below normal. To conclude, 1·3× PRP gel appears to progress chronic, non healing wounds in SCI patients into the granulation phase of healing quickly. Review of the literature shows these results may not be applied to all PRP preparations.

INTRODUCTION

Pressure ulcers and open wounds as a result of trauma are a well‐known complication of spinal cord injury (SCI). The incidence of pressure ulcers in acute SCI rehab and the prevalence of pressure ulcers in the community‐dwelling persons with SCI have been reported to be 33–40% (1). More than 50% of all persons with SCI are likely to develop a pressure ulcer during their lifetime (2). These wounds are typically stage 3 or 4 pressure ulcers that go through the full thickness of the skin and extend into or through subcutaneous muscle and fascia and/or to the bone (3).

Reported recurrence rates for pressure ulcers in those with SCI range from 31% to 79% (4). Myocutaneous flap procedures are often considered to close large open wounds quickly. Of those pressure ulcers that are closed by surgical myocutaneous flap procedures, recurrence rates range from 6% to 61% depending on the study and the presence of postoperative complications (1).

Owing to the risks associated with surgery, the high recurrence rates noted above, difficulty in obtaining plastic surgery consults and lack of operating room time, surgeons have raised the criteria for flap surgery consideration (5). In addition, many persons with SCI and wounds are poor candidates or non candidates for extensive surgery because of conditions such as advanced age, poor nutrition, smoking addiction, previous flap procedures affecting donor sites or other comorbid medical conditions. These criteria leave a large segment of these patients needing to heal large pressure ulcers with conservative means.

There are significant physiological differences in the tissues below the level of the SCI. These differences include intrinsic physiologic derangement (6), a reduced vascular response to loading, reduced muscular tone (7) and progressive loss of muscle bulk (7). Other differences include decreased blood flow, blood pressure and blood supply, decrease in enzymes of biosynthesis, inherent collagen catabolism, increase in urinary excretion of glycosaminoglycans and decreased fibronectin, a glycoprotein for fibroblast activity. When these individual deficits are considered in the light of the cascade of events needed to heal a wound, it becomes apparent that the person with SCI is naturally impaired at every step of the physiological process for wound healing (8).

With this as a background, there is a need to investigate alternative wound healing methods to incite these recalcitrant and life‐altering wounds in persons with SCI to progress towards closure.

PLATELET‐RICH PLASMA GEL

The use of autologous platelet‐derived growth factors in wound healing was first reported in peer‐reviewed literature in the mid‐1980s and early 1990s 9, 10. Since then, the use of platelet‐rich plasma (PRP) gel containing multiple growth factors and plasma proteins has been reported consistently in the literature for treating acute and chronic cutaneous wounds 11, 12, 13, 14, 15, 16, 17 and in sports medicine and orthopaedic surgery, plastic surgery and maxillofacial and dental surgery 15, 18. As a side note of importance in chronic wound care, but not necessarily SCI, pain reduction with the use of PRP has been noted by many authors 16, 18, 19, 20, 21.

Most PRP gels are prepared using some basic common steps, with many variations and additions to those steps. These variations can affect the effectiveness of the gel. A sample of autologous blood is centrifuged to separate the red blood cells from the plasma and its contents including platelets, fibrin, fibronectin and albumin. The platelets are stimulated to degranulate and release the growth factors, cytokines and chemokines they carry that are the necessary chemical signals to incite the steps of the wound‐healing process. At the same time the fibrinogen in the plasma is activated to form a fibrin matrix scaffold, resulting in a gel that varies from a thick syrup consistency to a malleable carrier for the growth factors. In the United States, all PRP is autologous, that is, it is made from the patient's own blood. The PRP used in this study is made and applied by the treating clinician using blood obtained from the patient in the same day as treatment.

Because of the importance of growth factors to all aspects of wound healing and the measurably decreased levels of growth factors in chronic wounds, topically applied growth factors in the form of PRP gel have been used to restart the healing process in chronic wounds 11, 14, 16, 17, 20, 22. A myriad of growth factors released by platelets regulate the orchestrated and complex events in normal wound healing. Although preparations of single growth factors have been tried in wound care, results have been mixed at best (23). PRP gels offer the benefit of supplying the full complement of growth factors that synergise to provide the body's normal healing capability (18).

Making PRP

There are many variations in the basic steps described above for making PRP. Variations in the making of PRP include centrifuge parameters of rotor speed and angulation, length of time of centrifugation (1–45 min), method of activation of platelets, ratio of blood to activation factor, post‐centrifugation blood components used, additional components added to the gel and the resulting concentration of platelets (1× baseline to 8× or more). Several authors have stated that much of the reported variability in wound healing outcomes following the use of PRP is likely because of the diversity of devices, methods and clinical strategies used to obtain and apply PRP‐derived products. These authors also note that these various preparation methods probably yield different PRPs, which probably yield different results in wound healing 24, 25, 26, 27. To date, there are no published studies comparing clinical outcomes between any two different PRP preparations.

Evidence for the use of PRP for healing chronic wounds

Methods using cryoprecipitates and frozen platelet lysates were ineffective for wound healing or showed marginal results at best 19, 28, 29, 30. Those methods with lower concentrations of platelets – one to three times baseline – showed more robust healing rates 14, 22 than those with higher concentrations of three to eight times baseline 11, 16, 17, 20. In a meta‐analysis, Martinez‐Zapata et al. found that combining the few high‐quality studies available showed a benefit to treating chronic wounds with PRP (31). These studies used PRP with lower concentrations of platelets.

The process (AutoloGel™ System, Cytomedix Inc., Gaithersburg, MD) used in this paper incorporates a high rate of spin for 1 min in a proprietary centrifuge designed to preserve the majority of the platelets in the plasma. All of the plasma from a small blood sample is used to access albumin, fibrin and fibronectin. None of the red blood cells are used. In this preparation, ascorbic acid is added to the PRP to scavenge the free radicals in the chronic wound bed, increase fibroblasts and new tissue deposition and stimulate the development of thicker and more organised collagen (32). Degranulation and the gelling process are accomplished with the addition of calcified thrombin. This is the only PRP method cleared by the Food and Drug Administration for use in chronic wounds (33). This method yields a PRP that is 1·3 times the concentration of platelets at baseline for that patient and is hereafter referred to as 1·3× PRP. This method has been shown in the literature to enhance wound healing 12, 13, 14.

Purpose

The purpose of this paper is to investigate the effect of PRP gel in improving chronic wounds in persons with SCI towards healing by examining all patients with SCI in a large case series.

METHODS

The subjects in this case series are those from a 200‐patient and 285‐wound case series collected in 39 sites from November 2008 to August 2010 who were identified as having a SCI. These sites were either evaluating 1·3× PRP for inclusion in their formulary or were regularly using the 1·3× PRP in their wound care program. Each of these sites had been trained in wound preparation, PRP preparation and application, post‐application dressing and recommended wound care between treatments.

Inclusion criteria for the use of 1·3× PRP were: (i) any open, cutaneous wound that the clinician determined was not progressing in healing, (ii) wounds that could have a majority clean wound bed just prior to application of the product, (iii) wounds without clinical signs and symptoms of active infection and (iv) patient with SCI. Exclusion criteria were: (i) malignancy in the wound bed, (ii) concurrent chemotherapy and (iii) active untreated wound infection.

All patients from these sites who met the criteria and had the product applied to the wound were included in the large‐study data analysis. No patient was excluded for any reason including those that often result in exclusion from studies, for example multiple serious comorbidities, drugs that inhibit wound healing, smoking or lab values such as albumin, pre‐albumin, haematocrit or haemoglobin below commonly accepted norms.

All patients with SCI who were treated with 1·3× PRP for chronic wounds in the 285‐wound set are included in this SCI data set. These patients came from 11 long‐term acute care hospitals (LTACs), two outpatient wound clinics, one home health care agency and one wound care equipment and service supplier who serviced patients in long‐term care and in home‐care settings (Table 1). The 20 subjects had 37 wounds among them and all wounds were treated with the 1·3× PRP. Only one wound per subject is included in this analysis. If a subject had more than one wound, the largest wound by volume was used.

Table 1.

Baseline subject and site data

Subjects and wounds
No. of patients	20
No. of wounds	20
Average age in years	49·2 (27–75)
Average wound duration in weeks (range)	79·4 (8–416)
Healthcare settings (# of sites)	# of wounds per setting
Long‐term acute care hospitals (10)	11
Outpatient wound centers (2)	2
Home health care (1)	1
Medical supplier in long‐term care hospitals and home health	6

During data collection, Health Insurance Portability and Accountability Act regulations (34) were followed regarding sharing de‐identified aggregate data for study purposes as per the National Institutes of Health Authorization for Research Uses and Disclosures. According to these regulations, Internal Review Board approval and individual patient informed consents were not required because (i) the data were gathered by clinicians during normal clinical care and (ii) all protected health information about the patients were de‐identified before analysis.

Data collection and analysis

As this study was not initiated as a study on those with SCI, typical information that would be gathered for an SCI study such as individual motor and sensory status, duration of injury, level of injury and completeness of injury measured by the American Spinal Injury Association (ASIA) Impairment Scale were not requested during the initiation of data collection.

Data were gathered prospectively during patient treatment and recorded in facility records or on a basic wound data form. Data gathered for the analysis included patient age, wound location, wound aetiology, treatment dates and wound measurements. Measurements included length, width, depth, undermining and sinus tracts/tunnels (ST/Ts). Undermining and ST/Ts were measured at 3:00, 6:00, 9:00 and 12:00 hours. The diversity of sites led to some diversity of measurement techniques, with the most prevalent being longest length, width and depth visible at each measurement point. In nearly all cases the same measurer did all measurements for that subject. If available, lab values for haemoglobin, haematocrit, platelet count, pre‐albumin and/or albumin were included in the data. In many settings lab values were not readily available.

The data were entered into a Microsoft Excel [Microsoft™, Bellevue, WA] spreadsheet that was programmed with formulas to calculate the following, as well as descriptive statistics:

• (i)Area – calculated using the formula for an ellipse: length × width × 0·7854. Reason: an ellipse is closer to a wound shape than a square or rectangle that would be described by simple length × width. This method of calculation has been used in peer‐reviewed wound care literature 35, 36.
• (ii)Volume – calculated using the formula (length × width × 0·7854) × depth.
• (iii)Undermining – total of the horizontal measurements from the deepest point of the undermining to the wound edge taken at 3:00, 6:00, 9:00 and 12:00 hours, and added together for a single linear total per wound.
• (iv)ST/Ts – total of the measurements from the deepest point of the tunnel to the closest wound edge taken at 3:00, 6:00, 9:00 and 12:00 hours and added together for a single linear total per wound.

The stage of the wound was not requested on the data collection sheets but full thickness (FT) versus partial thickness (PT) was requested.

Baseline data were taken at the time of the first 1·3× PRP treatment. If there was more than a 14‐day gap between recorded measurements, data were used only until the last measurement before the 14‐day break. Final data were taken as the first measurement after the last 1·3× PRP treatment. The timing of this measurement depended on when the clinician deemed it necessary to remove the dressing, between 2 and 6 days post‐application.

Treatment outcome was defined as percent of change for area, volume, undermining and ST/T and was calculated as (baseline measurement minus last assessment day measurement) divided by baseline measurement. Average actual change per day and per week was also calculated. In addition, the number of weeks of treatment and the number of treatments were recorded and averaged.

Those who responded positively to the therapy in each outcome area were examined as a group, called the responders. Analysis of this group indicates clinical relevance or the potential of a therapy to provide positive results in a group of patients, the magnitude of the response and the length of time and the number of treatments to achieve the response (37). This analysis assists the reader in determining clinically effective therapies.

RESULTS

Basic data on the subjects and wounds as well as the distribution of the sites are shown in Table 1. Average age of the patients was 49·2 years with a range of 27–75 years. Anecdotally, length of time post‐SCI injury ranged from 6 months to more than 40 years. Wound duration averaged 79·4 weeks or 1·5 years, with a wide range of 8–416 weeks (8 years). The median value for duration was 46 weeks, still nearly a 1 year time span.

To give a view of the range of wound severities in the cohort, the distribution of the depths of those wounds and the number of wounds on each body site are shown in Table 2. The wound stage might be estimated by wound depth, but staging a wound is dependent on the body tissue identified in the wound bed. Stage 2 pressure ulcers are partial thickness of the skin, while stage 3 and 4 wounds are through the full thickness of the skin and either to or into the subcutaneous tissues (38). In patients with thinner skin, a small depth measurement can actually be a full thickness wound. The total number of wounds in each of the depth ranges is shown, along with the number of wounds in that group indicated as ‘full thickness' on the data sheets. Fifteen of the 20 wounds were designated as full thickness, none were partial thickness and the remaining 5 did not indicate thickness. Wounds as shallow as 0·1 and 0·2 cm were designated as full thickness, corroborating that stage cannot be determined by depth because of the thinness of the skin of some patients and some body sites such as the heel.

Table 2.

Wound locations and depths (cm)

	0 to <0·5 cm	0·5 to <1·0 cm	1·0 to <2·0 cm	2·0 to <8·5 cm	Total wounds per body site
Ischial tuberosity	2	1	5	5	13
Sacrum/coccyx		1		2	3
Legs/feet	1	1	2		4
Total per wound depth (# specified as ‘full thickness')	3 (2)	3 (2)	7 (6)	7 (5)

In a mean of 4·0 treatments over 3·4 weeks, the wounds reduced by an average of 47·9% in area and 56·0% in volume. Of the 20 wounds, 4 (20%) had undermining, averaging 7·0 cm in cumulative length. Three of the wounds had ST/Ts, averaging 5·0 cm in depth. Those with undermining reduced an average of 31·4% using 3·5 treatments over 2·6 weeks. Those with ST/Ts reduced an average of 26·1% (range 20·0–33·3, SD 6·7) using 2·3 treatments over 1·5 weeks (Table 3).

Table 3.

Wound data outcome analysis

	Average baseline (range, SD)	Average percent reduction (range, SD)	Average change per week	Average change per day	# Weeks of treatment	# Treatments
Area (cm2)	25·6	47·9	4·3	·6	3·4	4·0
	(0·2–151·0, SD 41·4)	(−12·0 to 99·7, SD 31·4)
Volume (cm3)	53·4	56·0	10·7	1·5	3·4	4·0
	(−68·0 to 9·8, SD 67·9)	(−68·0 to 99·8, SD 44·1)
Undermining (cm) (4 of 20 wounds)	7·0 cm	31·4%	0·7	0·1	2·6	3·5
	(4·8–9·0, SD 2·1)	(−15·6 to 69·1, SD 37·4)
Sinus tracts/tunnels (cm) (3 of 20 wounds)	5·0 cm	26·1%	1·1	0·2	1·5	2·3
	(1·5–7·5)	(20·0–33·3, SD 6·7)

Eighteen of these 20 wounds were pressure ulcers. A comparison of these outcome results to the 142 wounds in the pressure ulcer group of the entire cohort of 285 wounds was performed and are written here as SCI results first followed by the comparable figure for the pressure ulcer group of the entire cohort in parentheses. Average percent area reduction was 47·9% (39·7%), average area reduction per day was 0·6 cm² (0·54 cm²), average percent volume reduction was 56·0% (52·4%), average volume reduction per day was 1·5 cm³ (2·1 cm³). While the SCI was treated on average 4·0 times over 3·4 weeks, the 142 pressure ulcers were treated on average 2·6 times over 2·0 weeks. Comparison of the undermining and ST/T data is not relevant because the number of subjects in this SCI group with either undermining or ST/Ts is so small.

Clinical relevance or the application of these results to the clinic setting is examined by analysing those who responded positively to the therapy (Table 4). In area and volume, 90% responded positively with an average reduction of 53·8% and 67·3% respectively. Volume and area reductions occurred in an average of 4·0 treatments over 3·4 weeks. Three of the four subjects (75%) with undermining responded positively and reduced 47% using an average 4·0 treatments over 2·8 weeks. While only three subjects had ST/Ts, all of them (100%) responded positively with an average reduction of 26·1%, using 1·5 treatments over 2·3 weeks.

Table 4.

Analysis of responders – clinical relevance – What is the likelihood that a wound will respond positively to the treatment? What kind of response might be expected?

	Percent of wounds positively responding	Average percent reduction of those positively responding	Average change per week	Average change per day	# Weeks of treatment	# Treatments
Area (cm2)	90%	53·8%	4·8	0·7	3·4	4·0
	18 of 20
Volume (cm3)	90%	67·3%	15·6	2·2	3·4	4·0
	18 of 20
Undermining (cm)	75%	47%	1·2	0·2	2·8	4·0
	3 of 4
Sinus tracts/tunnels (cm)	100%	26·1%	1·05	0·15	1·5	2·3
	3 of 3

Lab values were reported on only nine of the patients. Means for platelet count, 255·4 K (normal range 150–350), albumin, 3·2 g/dl (normal range 3·5–5·0), and pre‐albumin, 24·4 g/dl (normal range 16–35), were within normal ranges. However, means for haemoglobin of 11·4 g/dL (normal range for males 13–18) and for haematocrit of 34·6% (normal range for males 45–62) were below normal.

There were no reports of complications such as infection development or trauma to surrounding tissues. Although data concerning this point is not quantifiable, it is worth noting.

DISCUSSION

Chronic wounds differ substantially from acute wounds, including high bacterial load, low density of beneficial cytokines, high levels of inflammatory cytokines, high levels of matrix metalloproteinases (MMPs) thought to degrade extracellular matrix proteins and tissue and low levels of MMP inhibitors 23, 39. These physiological differences make chronic wounds more difficult to heal than acute wounds. Wounds on persons with SCI may be even more difficult to heal because of the physiological deficits that an SCI causes as discussed earlier in this article. Anecdotally, wounds in persons with SCI are frustrating for clinicians and patients alike as these wounds are traditionally chronic and slow to heal. A modality that shows a consistent positive response, especially in volume, undermining and ST/Ts would enhance care of these patients and may decrease costs by replacing less effective modalities. Unfortunately, the low number of subjects with undermining and ST/Ts prevents conclusions from being drawn for these parameters.

The majority of the wounds were full thickness stage 3 or 4 pressure ulcers. These severities are not unusual for wounds in persons with SCI (4). Pressure ulcers typically start in deeper tissues that are more susceptible to pressure. When damage is finally visible on the surface, the wound is often very deep but unknown to the patient who is typically insensate. These deep ulcers are slow to heal and so are long in duration.

Measured by both average and median statistics, the wounds included in this study were long‐standing, averaging 79·4 weeks or 1·5 years, with a median value of 46·0 weeks. The wounds on the low end of the duration range had a recorded duration of only 8 weeks, but had shown no signs of progressing towards closure with standard, evidence‐based wound care treatment.

As a group, these wounds improved rapidly with the 1·3× PRP treatment. By most standards, a 56% volume decrease in 3·4 weeks is a marked step forward from non healing. Time and financial investments are minimised when these results occur with only four applications.

This study group showed similar volume reduction results to the larger pressure ulcer group (56% versus 52·4%, 1·5 versus 2·1 cm³/day) but took longer (3·4 versus 2·0 weeks) and required slightly more treatments (4·0 versus 2·6). Visible and measurable results were robust. Curiously, area reduction in this SCI group was higher (average 47·9% versus 39·7%, 0.6 versus 0.54 cm²/day) than the larger group, but in the same slower timeframe.

Outcomes for outliers or those who were treated for extended time periods were analysed compared with the SCI group averages. The four outlier subjects were treated for an average of 8·5 weeks (range 7–10·4 weeks). However, their volume reduction at the group average 3·4 week treatment time was 87·7% (range 81·0–93·9) and average final volume reduction of 98·1%. It appears that most of the healing occurred in the first 3·4 weeks of treatment. This somewhat corroborates findings of Scevola et al. who reported that, in subjects with SCI and deep pressure ulcers, there was a statistically significant increase in the onset of the granulation phase of wound healing, the healing process was triggered faster and there was more healing in the first 2 weeks of treatment with allogenic PRP gel compared with ‘current best practice approach to chronic wound dressing protocol’(40).

Continuity of care and adherence to a complete wound care program optimises outcomes and a support surface is a key component of a wound care program for persons with SCI and wounds on the ischium, sacrum or coccyx. Appropriate support surfaces along with offloading and repositioning were used for all subjects in the LTACs, home health and medical supplier categories. The support surfaces used by the outpatient wound centre subjects are unknown, as is the adherence of these subjects to offloading and repositioning. For all subjects, the 1·3× PRP was used by knowledgeable wound care clinicians as part of a total program of wound bed preparation, nutrition and moisture balance.

One goal of clinical studies is to give an indication as to how relevant the modality being studied is to the clinical setting. Phrased as a question, ‘How likely is it that this will work on my patients?’ and ‘What is the response that might be expected?’ The positive response rates in Table 4 indicate that there is a very good chance (90%) that patients with SCI will respond positively and markedly to this therapy.

Much wound care literature, research and policy depends on visible wound area outcomes. In the clinic setting, wound care clinicians and patients know that initially it is more important to see healing in the wound depth affecting wound volume, than to see area reduction. The outcomes in this study portray this sequence, as wound volume showed more improvement than area, probably because of the relatively short 3·4‐week average treatment time. Had these patients been followed beyond this, area improvements may have caught up to the improvements in volume.

Low lab values have been linked to nutritional deficits and impairment of wound healing (41). The subjects in this study had lower than normal levels for haemoglobin and haematocrit, yet healing progressed in all wound parameters.

Treatment with the 1·3× PRP was not used to complete healing. In a real clinic setting more costly modalities are often used to improve the health of a wound and progress it towards healing, then treatment reverts to a less costly modality to continue the progress. This was the case with the use of the PRP. At the discretion of the treating clinician, this more costly modality was discontinued when the wound was well on the road to healing and a less costly modality was appropriate. In addition, the reimbursement structure in LTACs, comprising 10 of the 14 sites used for this study, covers an average 25 day length of stay, and most patients were lost to follow‐up after discharge. Because of this loss to follow‐up, it is unknown how many of the wounds reached full closure or how long or if the wounds treated in this study remained closed.

The method of making PRP used in this study results in a 1·3× concentration of platelets. All of the plasma, fibrin, fibrinogen and other plasma components are included in the gel and ascorbic acid is added in a proprietary formulation. The method is described earlier in this paper. Because variations in preparation yield different end products, these results cannot be applied to all PRP preparations. This 1·3× PRP gel formulation rebalances the chronic wound bed by clearing contaminating proteases and free radicals, providing the growth factors, cytokines and chemokines needed for cell growth and migration and providing the fibrin scaffold necessary for progression of granulation tissue. The inflammatory state is reduced and the wound is moved into the granulation phase of healing.

LIMITATIONS

One limitation of this study is the diversity of sites. It is impossible to standardise measurement techniques or treatment across 14 sites of care. The diversity of sites could also be considered a strength, as improvement was consistent across this wide variety of settings and differences in treatment method inherent between clinicians. Other data that could have been gathered to describe the subject population includes level of injury, length of time from SCI injury date, history of pressure ulcers and recurrence and previous modalities tried.

CONCLUSION

People with SCIs have major deficits in their wound healing cascade below the injury site. This causes pressure ulcers, which are prevalent in this population, to become chronic and result in costly health problems. The use of 1·3× PRP gel to treat these wounds appears to reverse this non healing trend and improve the wound into a strong healing phase in a timely fashion even in the presence of low haemoglobin and haematocrit.

ACKNOWLEDGEMENTS

This study was supported by Cytomedix Inc, Gaithersburg, MD. Dr Rappl is an employee of Cytomedix.