The regulation of wound healing, the cleaving of growth factor receptors, and the modification of the extracellular matrix are several functions of MMPs [9, 10]. The purpose of the present study was to assess variations of MMPs and TIMPs from activated, concentrated platelets from normal male blood donors. The major findings of the study included a significant increase in TIMP-2 and TIMP-4 concentrations for individuals aged over 30 compared to individuals 30 years of age and younger. MMP-2, MMP-9, TIMP-1, and ADAMTS13 did not demonstrate any statistically significant changes with age. The shift in the MMP:TIMP balance could potentially lead to a change in the cumulative effect of PRP therapies causing a decreased effectiveness of this modality in healing musculoskeletal pathologies as individuals age.
The use of concentrated, activated platelets or PRP remains popular in the clinical and operative setting despite mixed clinical results. Gardner et al.  performed a retrospective analysis including 61 patients undergoing total knee arthroplasty (TKA) with intraoperative use of PRP and compared outcomes of pain, motion, blood loss, and hospital stay to 37 control subjects. The PRP group had less blood loss as measured by preoperative and day 3 postoperative hemoglobin measurements (2.7-g/dL vs. 3.2-g/dL decrease, P = 0.026), decreased narcotic use while in hospital, achieved a higher range of motion prior to discharge, and were discharged an average of 1 day earlier compared to the control group.
Other studies for the therapeutic application of PRP demonstrated sustained improvement of symptoms for patients with lateral epicondylitis , decreased time to resume training for athletes after open repair of the Achilles tendon , and increased functional scores and decreased pain after rotator cuff repair . However, given the paucity of quality randomized controlled trials on this subject, the benefits are called into question upon further review by other authors. A meta-analysis reviewed 15 randomized controlled trials and five prospective cohort studies and found no clear clinical benefit of PRP . There was a trend of benefit favoring PRP use, but this was not statistically significant with wide confidence intervals. Varied clinical results have necessitated further basic science investigations to determine the optimal platelet concentration, preparation technique, and balance of cell signaling molecules to aid healing.
The effect of PRP is likely not due to the action of a single growth factor, cytokine, MMP, or TIMP, but instead a sum of synergistic effects of these dynamic factors during tissue remodeling and healing . MMPs are involved in wound healing and pathological conditions including osteoarthritis and rheumatoid arthritis . MMPs are proteases that degrade gelatin, collagen, elastin, aggrecan, osteonectin, cytokines, growth factors, and receptors . These molecules establish chemotactic gradients, regulate inflammation, and extravasation of leukocytes into injured tissue . MMP-9 (Gelatinase B) is directly implicated in regulating inflammation by modulating cell migration .
TIMPs have historically been thought of as inhibitors of MMPs with varying affinities for specific MMPs [9, 17, 18]. However, it has become clear that TIMP function may be more complex than previously estimated; in some cases, a TIMP may actually activate an MMP. For example, TIMP-2 activates MMP-2 (in complex with active MMP-14) when it binds to the hemopexin-like domain of MMP-2 . In contrast, TIMP-2 inhibits MMP-2 when it binds to the catalytic site of MMP-2 . TIMPs also affect cell proliferation independent of their inhibition effects on MMP activity . Kasper et al.  showed that MMPs and TIMPs likely induce mesenchymal stem cells in response to mechanical force at a fracture site. The authors argue that the balance of MMPs and TIMPs is likely the deciding factor, as opposed to the individual activity of any single bioactive molecule. Thus, these molecules must be considered to grasp the overall effects of PRP activity. The selected MMPs, TIMPs, and ADAMTS13 quantified in this study were chosen as a representative sample of these classes of molecules secondary to their release from platelets, biological effects, and reproducible, cost-effective commercial assays available to researchers.
The concomitant expression of MMPs and TIMPs has been documented in gene studies . The reason the protease and its inhibitor are simultaneously induced is for tight control of the extent of extracellular matrix degradation and remodeling . The correlation between MMP and TIMP concentrations in this study is likely for the same reason, but further investigation considering the independent functions of these molecules remains.
In this study, the authors observed a statistically significant increase in TIMP-2 and TIMP-4 with age (P = 0.04 and P = 0.04, respectively). This shift of balance in the protease:inhibitor ratio with age could alter overall tissue remodeling potential. This issue is made even more complex, given that TIMP-2 has multiple binding sites on MMP-2, at least one of which is required first for protease activation . Only after activation, would the inhibitory effects of TIMP-2 and other TIMPs targets be seen, via binding at the catalytic site. This mechanism of enzymatic activation by TIMPs, or pro-protein convertases, is best known with the previous example, but is presumed to occur for other MMPs as well . The authors hypothesize that an increased TIMP-2 and TIMP-4 concentration with age, as was demonstrated in this study, would lead to a change in MMP: TIMP equilibrium, causing an overall decrease of activity of the MMP family. This effect would also impact growth factor activity, chemokine activity, and the inflammation pathway. A decreasing trend in overall MMP activity with age as measured using a generic MMP substrate was observed. The cumulative effect of these components may result in the decreased efficacy of this therapeutic modality as a function of age.
Limitations of this study include the in vitro analysis of MMP and TIMP concentrations by ELISA. This is an appropriate technique to quantitate these biomolecules that would be given during therapeutic application of activated, concentrated platelets, but does not measure activity. No therapeutic intervention or patient outcome was evaluated in this study design. Given the male dominant military demographics of the donor population, insufficient female samples were collected to have adequate power for further analysis. Further studies should be conducted to include this population and perform clinical studies in conjunction with PRP composition analysis to correlate with patient outcome.
The selected biomarkers represent a small fraction of all biologically active molecules released from platelets and must be considered during interpretation of this study. The platelet proteome variation is known to change with advancing age . Therefore, other changes in biomarker expression in addition to the findings in this study may have a role in affecting healing. TIMP-1, TIMP-2, and TIMP-4 are known to reside in distinct patches separate from the α-granule that contains many molecules including Platelet Factor 4 (PF4), von Willebrand factor (VWF), clotting factors, and growth factors [23, 24]. The kinematics of release from these separate storage areas compared to the α-granule is not yet understood and could be a confounding factor.