Mitigation of experimental, chronic post-thoracotomy pain by preoperative infiltration of local slow-release bupivacaine microspheres.

BACKGROUND: Postoperative pain is treated incompletely and ineffectively in many circumstances, and chronic postoperative pain causes suffering and diminishes productivity. The objective of this project is to determine whether a recently developed slow-release formulation of bupivacaine was effective in reducing the experimental chronic postoperative pain. METHODS: In male Sprague-Dawley rats (250-300 g body weight), bupivacaine-releasing microspheres (MS-Bupi), containing 60 mg of bupivacaine base, were locally injected (MS-Bupi-L) 2 hours preoperatively into the subcutaneous compartment at the locus for experimental thoracotomy. Hypersensitivity to tactile stimulation was assessed by reductions in the threshold force required to induce a response to von Frey filaments (VFH) applied to the hairy back near the incision/retraction site. Pain behavior was assessed using a Qualitative Hyperalgesia Profile. Control groups included rats receiving the same dose of MS-Bupi but at a distant site on the back (MS-Bupi-D, testing for systemic drug actions) and rats receiving the same mass of microspheres with no drug (MS-Placebo) at the wound site. Rats were tested for 3 days before and 28 days (postoperative days [PODs]) after the procedure. Withdrawal threshold differences, which were the primary outcome measure, among all treatment groups were assessed by the Kruskal-Wallis test, after which pairwise comparisons were made by determining Wilcoxon-Mann-Whitney odds (WMWodds), with Bonferroni correction of the confidence intervals. RESULTS: Microsphere bupivacaine released near the incision reduced the chronic tactile allodynia after thoracotomy. The threshold values during PODs 14 to 28 were different among the 3 treatment groups when examined on PODs 14, 16, 18, 23, 25, and 28 but not on POD21 (P = 0.0603). WMWodds showed that threshold of the MS-Bupi-L group differed from those of the MS-Bupi-D and the MS-Placebo groups for all the tested PODs, whereas the thresholds of the MS-Bupi-D group never differed from those of the MS-Placebo group. Area-under-curve analysis for threshold reductions below baseline, using WMWodds, also showed a reduction during the entire 28 PODs that was greater for the MS-Bupi-L group compared with the MS-Placebo or MS-Bupi-D group. The incidence of intense pain scores by the Qualitative Hyperalgesia Profile analysis was observed in 7 of 8 rats in the MS-Placebo group and in 5 of 8 rats in the MS-Bupi-L group. CONCLUSIONS: Local slow release of bupivacaine subcutaneously from the MS-Bupi formulation suppresses postoperative mechanical hypersensitivity for ≥4 weeks after experimental thoracotomy. Systemic bupivacaine from this treatment has no effect on this hypersensitivity.

Local pathology and systemic serum bupivacaine after subcutaneous delivery of slow-releasing bupivacaine microspheres.

BACKGROUND: Prolonged local anesthesia, particularly desirable to minimize acute and chronic postoperative pain, has been provided by microspheres that slowly release bupivacaine (MS-Bup). In this study, we report on the systemic drug concentrations and the local dermatopathology that occur after subcutaneous injection of MS-Bup. METHODS: Rats (approximately 300 g) were injected under the dorsolumbar skin with MS-Bup containing 40 mg of bupivacaine (base) or with 0.4 mL of 0.5% bupivacaine-HCl (BupHCl; 1.78 mg bupivacaine). Blood was drawn, under sevoflurane anesthesia, at 10 minutes to 144 hours, and the serum analyzed for total bupivacaine by liquid chromatography-tandem mass spectrometry. In different animals, skin punch biopsies (4 mm) were taken at 1, 3, 7, 14, and 30 days after the same drug injections, sectioned at 5 µm, and stained with hematoxylin-eosin. Samples from skin injected with BupHCl, with MS-Bup suspended in carboxymethyl cellulose (MS-Bup.CMC), or in methyl cellulose (MS-Bup.MC) were compared with their respective drug-free controls (placebos). RESULTS: Serum bupivacaine reached a maximal average value (n = 8) of 194.9 ng/mL at 8 hours after injection of MS-Bup (95% upper prediction limit = 230.2 ng/mL), compared with the maximal average (n = 6) serum level of 374.9 ng/mL (95% prediction limit = 470.6 ng/mL) at 30 minutes after injection of BupHCl. Serum bupivacaine decreased to undetectable levels (<3.23 ng/mL) at 8 hours after BupHCl and was detectable at approximately 20% of the maximal value at 144 hours after MS-Bup injection. BupHCl injection resulted in moderate lymphocytic infiltration of skeletal muscle at 1 and 3 days. MS-Bup.CMC and placebo-CMC caused extensive infiltration of macrophages, lymphocytes, and some neutrophils at 1 to 7 days, whereas MS-Bup.MC and placebo-MC caused only mild inflammation. CONCLUSIONS: Subcutaneous administration of microspheres releasing bupivacaine results in lower blood levels lasting for much longer times than those from bupivacaine solution.

Prolonged amelioration of experimental postoperative pain by bupivacaine released from microsphere-coated hernia mesh.

BACKGROUND AND OBJECTIVES: Postoperative pain alters physiological functions and delays discharge. Perioperative local anesthetics are effective analgesics in the immediate 1- to 2-day postoperative period, but acute pain often lasts longer. The goal of this work was to develop a local anesthetic formulation adhering to an intraoperative implanted device that reduces pain for at least 3 days after surgery. METHODS: Six groups, each with 8 rats, were studied. In a control group (group I), one 1.2-cm-long incision of the skin was followed by blunt dissection to separate the skin away from the underlying tissues and closing with 2 sutures. In 3 of the treatment groups, the same surgical procedure was used, with the subcutaneous space formed by the blunt dissection lined with a 1-cm square patch of hernia mesh coated with poly lactide co-glycolic acid microspheres containing approximately 17 mg of bupivacaine (group II), no drug (placebo; group III), or bupivacaine free-base powder (group IV). Uncoated mesh implants (group V) served as a secondary control. A standard bupivacaine solution (0.4 mL, 0.5%; 2-mg dose) was infiltrated subcutaneously 30 minutes before the surgery and served as a standard control (group VI). Mechanosensitivity of the skin was tested by the local subcutaneous muscle responses to cutaneous tactile stimulation by von Frey hairs with forces of 4 g (for allodynia) and 15 g (for hyperalgesia) preoperatively and for 7 postoperative days. RESULTS: Control rats (group I) showed mechanohypersensitivity, indicative of postoperative allodynia and hyperalgesia, for all 7 postoperative days. Mechanohyperalgesia in rats that received mesh coated with bupivacaine-releasing microspheres (group II) was reduced during this period to 13% of control postoperative values (P < 0.001); mesh coated with bupivacaine base (group IV) reduced it by 50% (P = 0.034). The placebo mesh (group III) and uncoated mesh (group V) caused no significant reduction of mechanohypersensitivity, and bupivacaine solution infiltrated before the incision (group VI) reduced hypersensitivity for only approximately 2 hours, an overall insignificant effect. CONCLUSIONS: Bupivacaine slowly released for 72 hours from microspheres adsorbed to the hernia mesh significantly suppresses evoked postoperative hypersensitivity for at least 1 week and is more effective than a suspension of these microspheres or preoperative single-shot infiltration of bupivacaine.

Inhibition by local bupivacaine-releasing microspheres of acute postoperative pain from hairy skin incision.

BACKGROUND: Acute postoperative pain causes physiological deficits and slows recovery. Reduction of such pain by local anesthetics that are delivered for several days postoperatively is a desirable clinical objective, which is approached by a new formulation and applied in animal studies reported here. METHODS: We subcutaneously injected a new formulation of poly-lactic-co-glycolic acid polymer microspheres, which provides steady drug release for 96+ hours into rats at the dorsal region 2 hours before surgery. A single 1.2-cm-long skin incision was followed by blunt dissection of skin away from the underlying fascia, and closed by 2 sutures, followed by 14 days of testing. Microspheres containing 5, 10, 20, and 40 mg bupivacaine were injected locally 2 hours before surgery; bupivacaine-free microspheres were the vehicle control, and bupivacaine HCl solution (0.5%), the positive control. Mechanical sensitivity was determined by the frequency of local muscle contractions to repeated pokes with nylon monofilaments (von Frey hairs) exerting 4 and 15 g forces, testing, respectively, allodynia and hyperalgesia, and by pinprick. RESULTS: Injection of bupivacaine microspheres (40 mg drug) into intact skin reduced responses to 15 g von Frey hairs for 6 hours and to pinprick for 36 hours. Respective reductions from bupivacaine HCl lasted for 3 and 2 hours. Skin incision and dissection alone caused mechanical allodynia and hyperalgesia for 14 days. Microspheres containing 20 or 40 mg bupivacaine suppressed postoperative hypersensitivity for up to 3 days, reduced integrated allodynia (area under curve of response versus time) over postoperative days 1 to 5 by 51% ± 20% (mean ± SE) and 78% ± 12%, and reduced integrated hyperalgesia by 55% ± 13% and 64% ± 11%, for the respective doses. Five and ten milligrams bupivacaine in microspheres and the 0.5% bupivacaine solution were ineffective in reducing postoperative hypersensitivity, as were 40 mg bupivacaine microspheres injected contralateral to the incision. CONCLUSIONS: Significant suppression of postoperative pain by the slow-release bupivacaine preparation outlasts its anesthetic action on intact skin. These findings demonstrate preventive analgesia and indicate the importance of acute processes in the development of chronic postoperative pain.

Prolonged nerve block by microencapsulated bupivacaine prevents acute postoperative pain in rats.

BACKGROUND AND OBJECTIVES: To minimize acute postoperative pain, a new formulation of slowly released bupivacaine was developed. METHODS: Bupivacaine was microencapsulated at 60% (wt/wt) in poly-lactide-co-glycolide polymers and characterized for physicochemical properties and bupivacaine release kinetics. This formulation was injected around the rat sciatic nerve to produce an antinociceptive effect to toe pinch. Mechanical hyperalgesia following lateral plantar paw incision in rats was assessed for 7 to 14 days when the bupivacaine slow-release formulation was placed at the ipsilateral sciatic nerve and compared with the hyperalgesia that developed with various controls. RESULTS: Bupivacaine was released in vitro at a relatively constant rate over a period of ≈ 72 to 96 hours. Complete antinociception, shown as no response to toe pinch, lasted for 23 ± 7 hours, with a half-recovery time of 42 ± 8 hours after sciatic nerve injection of 0.4 mL of the microspheres delivering 34 mg of bupivacaine. Solutions of 0.5% (wt/vol) bupivacaine-HCl (0.1 mL) produced complete antinociception for less than 2 hours and recovery half-times of 2 hours. Postincisional mechanical hyperalgesia, shown by increased withdrawal responses to von Frey filaments, was absent for 24 hours and was lower than control for 96 hours, when the sciatic nerve was blocked by bupivacaine microspheres, whereas the 0.5% bupivacaine solution reduced postincisional pain for only 4 hours. CONCLUSIONS: Corresponding to its far greater functional blocking time, the microsphere-bupivacaine formulation was able to significantly reduce postoperative pain below control levels for up to 4 days. These findings of several days of postoperative pain relief, for an injectable formulation containing a single active agent, present an improved and potentially promising therapy to prevent acute pain after surgery.

Mitigation of ectopic calcification in osteopontin-deficient mice by exogenous osteopontin.

Ectopic calcification is a major cause of bioprosthetic heart valve failure. New therapeutic opportunities are offered by the growing understanding that ectopic calcification is an actively regulated process involving several key gene products. One of these products, osteopontin (OPN), is a glycosylated phosphoprotein previously shown to inhibit apatite crystal formation, induce carbonic anhydrase II, and promote mineral resorption. In this study, OPN-deficient mice (OPN-/-) were utilized as an in vivo model to stimulate the ectopic calcification of glutaraldehyde-fixed bovine pericardium (GFBP) tissue and to examine OPN delivery and structure-function relationships with respect to its anti-calcific activity. Significant calcification of GFBP tissue was obtained within 7 days of subcutaneous implantation in OPN-/- mice. Direct rescue of the calcification phenotype was achieved by the administration of exogenous recombinant rat, histidine-fused OPN (rat His-OPN) to the implant site via soluble injection (up to 72% mitigation achieved) or adsorption onto the implant materials (up to 91% mitigation achieved). Effects were specific, since neither fibronectin nor polyhistidine alone could mitigate calcification of GFBP. The maximum anti-calcific effect was achieved only when rat His-OPN was adequately phosphorylated and contained a functional arginine-glycine-aspartate (RGD) cell adhesive domain. Furthermore, CAII levels in host cells surrounding GFBP were greatest when phosphorylated, RGD-containing rat His-OPN was adsorbed. These data suggest that both physical inhibition, mediated by phosphorylation sites in OPN, as well as the induction of CAII and mineral regression, mediated by the RGD domain, contribute to the unique ability of OPN to mitigate ectopic calcification of bioprosthetic valve tissue.

Triglycidylamine crosslinking of porcine aortic valve cusps or bovine pericardium results in improved biocompatibility, biomechanics, and calcification resistance: chemical and biological mechanisms.

We investigated a novel polyepoxide crosslinker that was hypothesized to confer both material stabilization and calcification resistance when used to prepare bioprosthetic heart valves. Triglycidylamine (TGA) was synthesized via reacting epichlorhydrin and NH(3). TGA was used to crosslink porcine aortic cusps, bovine pericardium, and type I collagen. Control materials were crosslinked with glutaraldehyde (Glut). TGA-pretreated materials had shrink temperatures comparable to Glut fixation. However, TGA crosslinking conferred significantly greater collagenase resistance than Glut pretreatment, and significantly improved biomechanical compliance. Sheep aortic valve interstitial cells grown on TGA-pretreated collagen did not calcify, whereas sheep aortic valve interstitial cells grown on control substrates calcified extensively. Rat subdermal implants (porcine aortic cusps/bovine pericardium) pretreated with TGA demonstrated significantly less calcification than Glut pretreated implants. Investigations of extracellular matrix proteins associated with calcification, matrix metalloproteinases (MMPs) 2 and 9, tenascin-C, and osteopontin, revealed that MMP-9 and tenascin-C demonstrated reduced expression both in vitro and in vivo with TGA crosslinking compared to controls, whereas osteopontin and MMP-2 expression were not affected. TGA pretreatment of heterograft biomaterials results in improved stability compared to Glut, confers biomechanical properties superior to Glut crosslinking, and demonstrates significant calcification resistance.

Hyaluronic acid grafting mitigates calcification of glutaraldehyde-fixed bovine pericardium.

Pathologic calcification is the leading cause of the clinical failure of glutaraldehyde-fixed bovine pericardium used in bioprosthetic valves. A novel surface modification of glutaraldehyde fixed bovine pericardium was carried out with high molecular weight hyaluronic acid (HA). HA was chemically modified with adipic dihydrazide (ADH) to introduce hydrazide functional groups onto the HA backbone. Glutaraldehyde-fixed bovine pericardium (GFBP) was modified by grafting this HA to the free aldehyde groups on the tissue via the hydrazide groups. Following a 2-week subcutaneous implantation in osteopontin (OPN)-null mice, the calcification of HA-modified bovine pericardium was drastically reduced (by 84.5%) compared to positive controls (tissue without HA-modification) (p = 0.005). The calcification-mitigating effect of HA surface modification was also confirmed by microscopic analysis of explanted tissue stained with Alizarin Red S for calcium.