Histologic Evaluation of Osseous Regeneration Following Combination Therapy With Platelet-Rich Plasma and Bio-Oss in a Rat Calvarial Critical-Size Defect Model.

Platelet-rich plasma (PRP) is an autogenous source of growth factors shown to facilitate human bone growth. Bio-Oss, an osteoconductive xenograft, is used clinically to regenerate periodontal defects, restore dental alveolar ridges, and facilitate sinus-lift procedures. The purpose of this study was to analyze whether a combination of PRP and Bio-Oss would enhance bone regeneration better than either material alone. PRP and/or Bio-Oss were administered in an 8-mm critical-size defect (CSD) rat calvarial model of bone defect between 2 polytetrafluoroethylene membranes to prevent soft tissue incursion. Eight weeks after the induction of the CSD, histologic sections were stained with hematoxylin and eosin stain and analyzed via light microscopy. Qualitative analyses revealed new bone regeneration in all 4 groups. The Bio-Oss and PRP plus Bio-Oss groups demonstrated greater areas of closure in the defects than the control or PRP-only groups because of the space-maintaining ability of Bio-Oss. The groups grafted with Bio-Oss showed close contact with new bone growth throughout the defects, suggesting a stronger graft. The use of PRP alone or in combination with Bio-Oss, however, did not appear to enhance osseous regeneration at 8 weeks. Areas grafted with Bio-Oss demonstrated greater space-maintaining capacity than controls, and PRP was an effective vehicle for placement of the Bio-Oss. However, at 8 weeks this study was unable to demonstrate a significant advantage of using PRP plus Bio-Oss over using Bio-Oss alone.

Gender and age differences in growth factor concentrations from platelet-rich plasma in adults.

The use of platelet-rich plasma (PRP) to facilitate healing of orthopedic-related injuries has gained popularity; however, the clinical benefits are not consistent. Differences may result from variations in growth factor (GF) levels in normal populations. The purpose of this study was to determine if GF levels present in activated PRP preparations differed by gender and age (≤ 25 versus >25 years) in a healthy population (N = 102). All GFs analyzed (epidermal growth factor [EGF], hepatocyte growth factor [HGF], insulin growth factor-1 [IGF-1], platelet-derived growth factor-AB [PDGF-AB], platelet-derived growth factor-BB [PDGF-BB], transforming growth factor beta-1 [TGFß-1], and vascular endothelial growth factor) had higher levels for females and for those ≤ 25 years old. Of the GFs tested, four of seven were significantly higher (p < 0.05) for females (EGF, HGF, IGF-1, PDGF-BB), the most significant being IGF-1 (female, 85.0; male, 69.3 ng/mL; p < 0.01). Five of seven GFs achieved significance (p < 0.05) for people ≤ 25 years old (EGF, IGF-1, PDGP-AB, PDGF-BB, and TGFß-1), with IGF and PDGF-AB achieving p < 0.001 (≤ 25 years, 85.1; >25 years, 56.8, and ≤ 25 years, 7.66; >25 years, 5.77 ng/mL, respectively). Finally, for both genders, most of the GFs were positively correlated with all GFs. This study demonstrated that both age and gender account for variations in specific GFs present in PRP, and this may partially explain some of the inconsistent results of PRP clinical trials.

Variance of matrix metalloproteinase (MMP) and tissue inhibitor of metalloproteinase (TIMP) concentrations in activated, concentrated platelets from healthy male donors.

BACKGROUND: The use of autologous blood concentrates, such as activated, concentrated platelets, in orthopaedic clinical applications has had mixed results. Research on this topic has focused on growth factors and cytokines, with little directed towards matrix metalloproteinases (MMPs) which are involved in post-wound tissue remodeling. METHODS: In this study, the authors measured the levels of MMP-2, MMP-9 and a disintegrin and metalloproteinase with a thrombospondin type 1 motif, member 13 (ADAMTS13), in activated platelets derived from blood of healthy, male volunteers (n = 92), 19 to 60 years old. The levels of the natural inhibitors of these proteases, tissue inhibitor of metalloproteinase 1 (TIMP-1), TIMP-2 and TIMP-4 were also assessed. RESULTS: Notably, there was no significant change in concentration with age in four of six targets tested. However, TIMP-2 and TIMP-4 demonstrated a statistically significant increase in concentration for subjects older than 30 years of age compared to those 30 years and younger (P = 0.04 and P = 0.04, respectively). CONCLUSION: TIMP-2 and TIMP-4 are global inhibitors of MMPs, including MMP-2 (Gelatinase A). MMP-2 targets native collagens, gelatin and elastin to remodel the extracellular matrix during wound healing. A decreased availability of pharmacologically active MMP-2 may diminish the effectiveness of the use of activated, concentrated platelets from older patients, and may also contribute to longer healing times in this population.

Calpain inhibitor attenuated optic nerve damage in acute optic neuritis in rats.

Optic neuritis (ON), which is an acute inflammatory autoimmune demyelinating disease of the central nervous system (CNS), often occurs in multiple sclerosis (MS). ON is an early diagnostic sign in most MS patients caused by damage to the optic nerve leading to visual dysfunction. Various features of both MS and ON can be studied following induction of experimental autoimmune encephalomyelitis (EAE), an animal model of MS, in Lewis rats. Inflammation and cell death in the optic nerve, with subsequent damage to the retinal ganglion cells in the retina, are thought to correlate with visual dysfunction. Thus, characterizing the pathophysiological changes that lead to visual dysfunction in EAE animals may help develop novel targets for therapeutic intervention. We treated EAE animals with and without the calpain inhibitor calpeptin (CP). Our studies demonstrated that the Ca(2+)-activated neutral protease calpain was upregulated in the optic nerve following induction of EAE at the onset of clinical signs (OCS) of the disease, and these changes were attenuated following treatment with CP. These reductions correlated with decreases in inflammation (cytokines, iNOS, COX-2, and NF-κB), and microgliosis (i.e. activated microglia). We observed that calpain inhibition reduced astrogliosis (reactive astroglia) and expression of aquaporin 4 (AQP4). The balance of Th1/Th2 cytokine production and also expression of the Th1-related CCR5 and CXCR3 chemokine receptors influence many pathological processes and play both causative and protective roles in neuron damage. Our data indicated that CP suppressed cytokine imbalances. Also, Bax:Bcl-2 ratio, production of tBid, PARP-1, expression and activities of calpain and caspases, and internucleosomal DNA fragmentation were attenuated after treatment with CP. Our results demonstrated that CP decreased demyelination [loss of myelin basic protein (MBP)] and axonal damage [increase in dephosphorylated neurofilament protein (de-NFP)], and also promoted intracellular neuroprotective pathways in optic nerve in EAE rats. Thus, these data suggest that calpain is involved in inflammatory as well as in neurodegenerative aspects of the disease and may be a promising target for treating ON in EAE and MS.

Calpain inhibition attenuated morphological and molecular changes in skeletal muscle of experimental allergic encephalomyelitis rats.

Muscle weakness and atrophy are important manifestations of multiple sclerosis (MS). To investigate the pathophysiological mechanisms of skeletal muscle change in MS, we induced experimental autoimmune encephalomyelitis (EAE) in Lewis male rats and examined morphological and molecular changes in skeletal muscle. We also treated EAE rats with calpepetin, a calpain inhibitor, to examine its beneficial effects on skeletal muscle damage. Morphological changes in muscle tissue of EAE rats included smaller and irregularly shaped muscle fibers and fibrosis. Western blot analysis demonstrated increased calpain:calpastatin ratio, inflammation-related transcription factors (nuclear factor-κB:inhibitor of κB α ratio), and proinflammatory enzymes (cyclooxygenase-2). TUNEL-positive myonuclei in skeletal muscle cells of EAE rats indicated cell death. In addition, markers of apoptotic cell death (Bax:Bcl-2 ratio and caspase-12 protein levels) were elevated. Expression of muscle-specific ubiquitin ligases (muscle atrophy F-box and muscle ring finger protein 1), was upregulated in muscle tissue of EAE-vehicle animals. Both prophylactic and therapeutic treatment with calpeptin partially attenuated muscle changes noted in EAE animals. These results indicate that morphological and molecular changes including apoptotic cell death and protein breakdown develop in skeletal muscle of EAE animals and that these changes can be reversed by calpain inhibition.

Calpain inhibition attenuates apoptosis of retinal ganglion cells in acute optic neuritis.

PURPOSE: Optic neuritis (ON), inflammation of the optic nerve, is strongly associated with the pathogenesis of multiple sclerosis (MS) and is initiated by the attack of autoreactive T cells against self-myelin antigens, resulting in demyelination, degeneration of retinal ganglion cells (RGCs), and cumulative visual impairment. METHODS: Experimental autoimmune encephalomyelitis (EAE) was induced in Lewis rats on day 0, and animals received daily intraperitoneal injections of calpain inhibitor (calpeptin) or vehicle from day 1 until killed. Retinal cell death was analyzed by DNA fragmentation, and surviving ganglion cells were quantified after double labeling of retinal tissue with TUNEL and Brn3a. The expression of apoptotic and inflammatory proteins was determined by Western blotting. RESULTS: It was demonstrated that calpain inhibition downregulates expression of proapoptotic proteins and the proinflammatory molecule nuclear factor-kappa B (NF-κB) in the retina of Lewis rats with acute EAE. Immunofluorescent labeling revealed that apoptotic cells in the RGC layer of vehicle-treated EAE animals were Brn3a positive, and a moderate dose of calpeptin dramatically reduced the frequency of apoptotic RGCs. CONCLUSIONS: These results suggest that calpain inhibition might be a useful supplement to immunomodulatory therapies such as corticosteroids in ON, due to its neuroprotective effect on RGCs.

Calpeptin attenuated inflammation, cell death, and axonal damage in animal model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE) is an animal model for studying multiple sclerosis (MS). Calpain has been implicated in many inflammatory and neurodegenerative events that lead to disability in EAE and MS. Thus, treating EAE animals with calpain inhibitors may block these events and ameliorate disability. To test this hypothesis, acute EAE Lewis rats were treated dose dependently with the calpain inhibitor calpeptin (50-250 microg/kg). Calpain activity, gliosis, loss of myelin, and axonal damage were attenuated by calpeptin therapy, leading to improved clinical scores. Neuronal and oligodendrocyte death were also decreased, with down-regulation of proapoptotic proteins, suggesting that decreases in cell death were due to decreases in the expression or activity of proapoptotic proteins. These results indicate that calpain inhibition may offer a novel therapeutic avenue for treating EAE and MS.

Activation of calpain and caspase pathways in demyelination and neurodegeneration in animal model of multiple sclerosis.

Experimental autoimmune encephalomyelitis (EAE), a widely recognized animal model of multiple sclerosis (MS), is highly useful for studying inflammation, demyelination, and neurodegeneration in the central nervous system (CNS). EAE exhibits many similarities with MS, which is a chronic inflammatory disease affecting CNS white matter in humans. Various studies have indicated that EAE is a particularly useful animal model for understanding both the mechanisms of immune-mediated CNS pathology and also the progressive clinical course of MS. Demyelination and axonal dysfunction have previously been shown in MS and EAE but current evidences indicate that axonal damage and neuron death also occur, demonstrating that these diseases harbor a neurodegenerative component. Recent studies also have shown that the activation of calpain and caspase pathways contribute to the apoptotic death of oligodendrocytes and neurons, promoting the pathological events leading to neurological deficits. Apoptosis is involved in the disease-regulating as well as in the disease-promoting processes in EAE. This review discusses the major involvement of calpain and caspase pathways in causing demyelination and neurodegeneration in EAE animals.

Time-dependent increases in protease activities for neuronal apoptosis in spinal cords of Lewis rats during development of acute experimental autoimmune encephalomyelitis.

Multiple sclerosis (MS) is characterized by axonal demyelination and neurodegeneration, the latter having been inadequately explored in the MS animal model experimental autoimmune encephalomyelitis (EAE). The purpose of this study was to examine the time-dependent correlation between increased calpain and caspase activities and neurodegeneration in spinal cord tissues from Lewis rats with acute EAE. An increase in TUNEL-positive neurons and internucleosomal DNA fragmentation in EAE spinal cords suggested that neuronal death was a result of apoptosis on days 8-10 following induction of EAE. Increases in calpain expression in EAE correlated with activation of pro-apoptotic proteases, leading to apoptotic cell death beginning on day 8 of EAE, which occurred before the appearance of visible clinical symptoms. Increases in calcineurin expression and decreases in phospho-Bad (p-Bad) suggested Bad activation in apoptosis during acute EAE. Increases in the Bax:Bcl-2 ratio and activation of caspase-9 showed the involvement of mitochondria in apoptosis. Further, caspase-8 activation suggested induction of the death receptor-mediated pathway for apoptosis. Endoplasmic reticulum stress leading to caspase-3 activation was also observed, indicating that multiple apoptotic pathways were activated following EAE induction. In contrast, cell death was mostly a result of necrosis on the later day (day 11), when EAE entered a severe stage. From these findings, we conclude that increases in calpain and caspase activities play crucial roles in neuronal apoptosis during the development of acute EAE.

A role for calpain in optic neuritis.

Optic neuritis (ON) is one of the most commonly presenting symptoms of multiple sclerosis (MS), which is a neurodegenerative disease of the central nervous system (CNS) thought to be caused by an attack on myelin by autoreactive T cells and other immune cells. Experimental autoimmune encephalomyelitis (EAE) is a widely used model for MS and ON, which are characterized by demyelination, axonal damage, and neuronal death. The mechanisms of neurodegeneration are unclear; however, the calcium (Ca2+)-dependent neutral protease calpain is thought to be involved. The focus of this article is to summarize the evidence suggesting that calpain plays a role in the development of EAE-ON in Lewis rats.

Upregulation of calpain correlates with increased neurodegeneration in acute experimental auto-immune encephalomyelitis.

Although calpain up-regulation is well established in experimental auto-immune encephalomyelitis (EAE), a link between increased calpain expression and activity and neurodegeneration has not been examined. Therefore, spinal cord tissue from Lewis rats with EAE was examined to test the hypothesis that increased calpain expression in neurons would correlate with increased cell death and axonal damage in a time-dependent manner following EAE induction. We found that increased calpain expression in EAE corresponded to increased TUNEL-positive neurons and to increased expression of dephosphorylated neurofilament protein, markers of cell death and axonal degeneration, respectively. An increase in internucleosomal DNA fragmentation in EAE spinal cord suggested that cell death was, at least partially, due to apoptosis. Axonal damage was further demonstrated in EAE spinal cord compared with control via morphological analysis, revealing granular degeneration of filament and microtubule integrity, loss of myelin, and mitochondrial damage. Calcium (Ca2+) influx, which is required for calpain activation, was also increased in EAE spinal cord. From these findings, we conclude that increases in Ca2+-induced calpain activity may play a crucial role in neurodegeneration in acute EAE.

Calpain inhibitor prevented apoptosis and maintained transcription of proteolipid protein and myelin basic protein genes in rat spinal cord injury.

Spinal cord injury (SCI) is associated with progressive neurodegeneration and dysfunction. Multiple cellular and molecular mechanisms are involved in this pathogenesis. In particular, the activation of proteases following trauma can cause apoptosis in the spinal cord. Calpain, a calcium-dependent cysteine protease, plays a major role in apoptosis following trauma. We identified apoptosis and decrease in transcription of the genes for proteolipid protein (PLP) and myelin basic protein (MBP) in five 1-cm long spinal cord segments (S1, distant rostral; S2, near rostral; S3, lesion; S4, near caudal; and S5, distant caudal) 24 h after induction of SCI (40 g.cm force) in rats by weight-drop method. Sham rats underwent laminectomy and did not receive injury. Internucleosomal DNA fragmentation occurred prominently in the lesion (S3), moderately in near segments (S2 and S4), and slightly in distant segments (S1 and S5) of injured rats, indicating the occurrence of apoptosis in the lesion and penumbra. Levels of transcription of PLP and MBP were reduced highly in the lesion and moderately in near segments, suggesting that apoptotic loss of cells impaired biosynthesis of two important structural components of myelin. Immediate administration of the calpain inhibitor E-64-d (1 mg/kg) to injured rats prevented apoptosis and restored transcription of these genes, indicating the therapeutic efficacy of calpain inhibitor for treatment of SCI.