Effective wound healing in streptozotocin-induced diabetic rats by adipose-derived stromal cell transplantation in plasma-gel containing fragmin/protamine microparticles.

We investigated the effectiveness of the application of inbred adipose-derived stromal cells (IR-ASCs) in high inbred rat plasma (IRP) (6%)-Dulbecco modified Eagle medium (DMEM) gel with fragmin/protamine microparticles (F/P MPs) (IR-ASCs + IRP-DMEM gel + F/P MPs) on wound healing in streptozotocin-induced diabetic rats. F/P MPs have previously been used as a cell carrier for IR-ASCs in inbred Fisher 344 rats and for preservation and controlled release of various cytokines in IRP-DMEM gel. We applied IR-ASCs + IRP-DMEM gel + F/P MPs to full-thickness skin excisions on the backs of the diabetic rats. The statistical significance of wound closure was evaluated on postwounding days 3, 7, 10, and 14, and the skin area surrounding the wound was removed for histological examination on days 7 and 14. The wound closure rate and histological examination of wounds treated with IR-ASCs + IRP-DMEM gel + F/P MPs demonstrated significantly advanced epithelialization, capillary formation, and granulation tissue formation. When DiI-labeled IR-ASCs + IRP-DMEM gel + F/P MPs were applied to full-thickness skin wounds on the backs of the diabetic rats, histological observation at 2 weeks showed appearances of both DiI-labeled granulation tissue and CD31-immunostained microvessels in the transplant areas. A portion of the transplanted IR-ASCs + IRP-DMEM gel + F/P MPs had been taken up into the granulation tissues to promote wound healing. Thus, IR-ASCs + IRP-DMEM gel + F/P MPs were effective for repairing healing-impaired wounds such as those arising in the diabetic rats.

Delivery system for autologous growth factors fabricated with low-molecular-weight heparin and protamine to attenuate ischemic hind-limb loss in a mouse model.

Frozen and thawed platelet-rich plasma (PRP) contains high concentrations of various growth factors, such as fibroblast growth factor (FGF)-2, vascular endothelial growth factor, and hepatocyte growth factor. We previously reported that low-molecular-weight heparin/protamine microparticles (LH/P MPs) are useful as biodegradable carriers for the controlled release of FGF-2. In this study, we examined the ability of PRP/LH/P MPs to prevent limb loss in an induced ischemic hind-limb model that used adult BALB/c-nu/nu male mice. One day after inducing ischemia, intramuscular injections of a PRP/LH/P MPs solution were administered into several sites of the ischemic hind limb. Seven days and onward after the injections, the PRP/LH/P MPs-treated and PRP-treated groups recovered from ischemia, as reflected by the improved oxygen saturation. In the PRP-treated group, however, the level of recovery of oxygen saturation after ischemia decreased after 14 days. From the 21st day onward, there was a significant difference between those two groups. In the LH/P MPs-treated group, a partial recovery occurred only in the early period. The saline-treated group (i.e., the control) and the noninjection group (i.e., ischemia only) exhibited no recovery. The limb survival rate at 1 year in the ischemia-induced mice injected with PRP/LH/P MPs was approximately 25 % (two of eight mice) but was absent in the other groups.

Radiation-induced Skin Injury on the Upper Arm Following Cardiac Interventional Radiology: A Review and Case Report.

Cardiac interventional radiology (IVR) has gained popularity since the late 1980s. An extensive search of literature published from 1996 to 2009 identified 78 reports regarding radiation-induced skin injuries (RISI) following cardiac IVR. The authors present a case report and a review of the relevant literature. A 59-year-old man underwent percutaneous transluminal coronary angioplasty (PTCA). The estimated cumulative skin dose was 11.8 Gy. The patient discovered erosions on the right upper arm 2 months after PTCA. The lesion did not resolve with conservative treatment, and evolved into an intractable skin ulcer. The authors surgically removed the lesion and reconstructed the defect using an anterolateral thigh (ALT) flap. Radiation-induced skin injuries following PTCA usually develop on the back, and rarely on the upper arm. Radiation ulceration commonly requires surgical intervention. Physicians should be aware of the possibility of RISI, and thus, take measures to minimize exposure of patients and staff to radiation. .

PRP&F/P MPs improved survival of dorsal paired pedicle skin flaps in rats.

BACKGROUND: Skin flap necrosis is a problem encountered postoperatively. The purpose of this study was to evaluate the effects of platelet-rich plasma containing fragmin/protamine microparticles (PRP&F/P MPs) on viability in a rat dorsal paired pedicle skin (DPPS) flap. MATERIALS AND METHODS: Two symmetrical adjoining rectangular flaps (8 × 2 cm each) were drawn on the rat dorsum. Two days after PRP&F/P MPs-, PRP-, F/P MPs-, and saline (control)-injections (n = 8 each), flaps were elevated as a random pattern flap without the lateral thoracic, posterior intercostal, and deep circumflex iliac vessels. The flaps were immediately sutured back and the flap survival area was measured 7 d after flap elevation. RESULTS: The flap survival rate in PRP&F/P MPs-injected groups (73.1% ± 4.2%) was significantly higher than those in PRP (64.9% ± 4.0%), F/P MPs (59.4 ± 4.5%), and control (61.2% ± 4.2%) groups. Histologic observation of the flaps showed survived thick granulation tissue and neovascularization in PRP&F/P MPs-injected groups. CONCLUSIONS: When PRP&F/P MPs are administered 2 d before the flap elevation, the improved flap survivals are observed. The pre-injection of PRP&F/P MPs may thus represent a promising treatment to prevent skin flap necrosis in reconstructive surgery.

Enhanced effect of platelet-rich plasma containing a new carrier on hair growth.

BACKGROUND: Treatments for alopecia are in high demand, but not all are safe and reliable. Dalteparin and protamine microparticles (D/P MPs) can effectively carry growth factors (GFs) in platelet-rich plasma (PRP). OBJECTIVE: To identify the effects of PRP-containing D/P MPs (PRP&D/P MPs) on hair growth. METHODS & MATERIALS: Participants were 26 volunteers with thin hair who received five local treatments of 3 mL of PRP&D/P MPs (13 participants) or PRP and saline (control, 13 participants) at 2- to 3-week intervals and were evaluated for 12 weeks. Injected areas comprised frontal or parietal sites with lanugo-like hair. Experimental and control areas were photographed. Consenting participants underwent biopsies for histologic examination. RESULTS: D/P MPs bind to various GFs contained in PRP. Significant differences were seen in hair cross-section but not in hair numbers in PRP and PRP&D/P MP injections. The addition of D/P MPs to PRP resulted in significant stimulation in hair cross-section. Microscopic findings showed thickened epithelium, proliferation of collagen fibers and fibroblasts, and increased vessels around follicles. CONCLUSION: PRP&D/P MPs and PRP facilitated hair growth but D/P MPs provided additional hair growth. The authors have indicated no significant interest with commercial supporters.

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with hydrosheets composed of chitin/chitosan, fucoidan, and alginate as wound dressings.

To create a moist environment for rapid wound healing, a hydrosheet composed of alginate, chitin/chitosan, and fucoidan (ACF-HS) has been developed as a functional wound dressing. The aim of this study was to evaluate the accelerating effect of ACF-HS on wound healing for rat mitomycin C-treated healing-impaired wounds. Full-thickness skin defects were made on the back of rats and mitomycin C was applied onto the wound for 10 minutes to prepare a healing-impaired wound. After thoroughly washing out the mitomycin C, ACF-HS was applied to the healing-impaired wounds. The rats were later euthanized and histological sections of the wounds were prepared. The histological examinations showed significantly advanced granulation tissue and capillary formations in the healing-impaired wounds treated with ACF-HS on days 7 and 14, in comparison with that in alginate fiber (Kaltostat), hydrogel wound dressing (DuoACTIVE), and nontreatment (negative control). Furthermore, in cell culture studies, ACF-HS-absorbed serum and fibroblast growth factor-2 was found to be proliferative for fibroblasts and endothelial cells, respectively, and ACF-HS-absorbed serum was found to be chemoattractive for fibroblasts. However, our results may not be strictly comparable with general healing-impaired wound models in humans because of the cell damage by mitomycin C. In addition, more biocompatibility studies of fucoidan are essential due to the possibility of renal toxicity.

Platelet-rich plasma (PRP) promotes survival of fat-grafts in rats.

This study evaluated the effects of platelet-rich plasma (PRP) on resorption and adipocyte survival in autologous fat-graft of rats prepared with isogenous PRP. Fat grafts prepared without PRP (control group) became united to the tissue adjacent to the implantation site and were significantly resorbed from 30 days. On the other hand, fat grafts prepared with PRP (PRP group) demonstrated little resorption from 30 to 120 days and appeared pink, had a soft, supple feel, and were easily compressible. Histologic sections of grafts in the control and PRP groups at 10 days exhibited similar consolidation of the grafted tissue, which contained morphologically normal adipocytes with different degrees of granulation and capillary formation. From 20 days normal adipocytes were obviously decreased in the control group, while the PRP group demonstrated increased granulation tissue and capillary formation and good maintenance of normal adipocytes for at least 120 days.

Intramedullary fixation with bioabsorbable and osteoconductive hydroxyapatite/poly-L-lactide threaded pin in digital replantation.

In digital replantation, Kirschner wire (K-wire) fixation has commonly been used for osteosynthesis. On the other hand, K-wires are often obtrusive because of protrusion from the replanted digit. We describe a case series treated using hydroxyapatite/poly-L-lactide (HPLLA) threaded pins, which are not only bioabsorbable, but also osteoconductive, for osteosynthesis in crushed amputation or comminuted fracture, including distal phalanx amputation. Using an HPLLA threaded pin, 10 digital replantations were performed between July 2016 and April 2018. The precisely cut pin is manually pushed into the fracture site as an intramedullary nail. The pin is first pushed into the distal site, and then into the proximal site, after drilling with a K-wire of the same diameter as the pin. All amputations were crush type, and levels of amputations were Tamai zone I in three cases, zone II in two and zone III in five. Eight of the 10 digits survived. The two digits that did not survive showed venous insufficiency. Bone union of the eight digits was successfully obtained after 9-19 weeks (median 12 weeks). No adverse events occurred such as distortion of the pin, infection or foreign body reaction. The HPLLA threaded pin may be an adequate device to fix fractures in digital replantation.

A case of mediastinitis with an exposed artificial blood vessel that was associated with right ventricular wall damage during treatment.

We report a serious case of right ventricular wall damage during mediastinitis treatment, which was successfully treated with negative-pressure wound therapy with continuous instillation (NPWT-CI).

Appropriate Use of Intravenous Unfractionated Heparin after Digital Replantation: A Randomized Controlled Trial Involving Three Groups.

BACKGROUND: The purpose of this study was to clarify the appropriate use of unfractionated heparin as an anticoagulation agent after digital replantation. METHODS: This study was a prospective, randomized, single-blind, blinded-endpoint method, three-arm, parallel-group, controlled clinical trial conducted at a single institution. A total of 88 patients (101 fingers) following digital amputation and subsequent repair by anastomosis of both arteries and veins were randomly allocated into three groups: (1) control group (no heparin dose), (2) low-dose heparin group (10,000 IU/day), and (3) high-dose heparin group (start at 15,000 IU/day, then adjust the dose to achieve an activated partial thromboplastin time of 1.5 to 2.5 times the baseline). The outcomes were assessed regarding the proportion of success at 2 weeks after replantation of amputated digits, total or partial necrosis, and occurrence of complications. RESULTS: No significant differences were found among the three groups, except for complications of congestion. The odds ratio of the heparin group compared with the control group for a success proportion was 5.40 (95 percent CI, 0.85 to 34.20; p = 0.027) in subjects aged 50 years or older. Significant elevations of activated partial thromboplastin time, aspartate transaminase, and alanine aminotransferase occurred in high-dose heparin groups on day 7. CONCLUSION: Unfractionated heparin administration is considered effective for patients aged 50 years or older, although the routine use of unfractionated heparin is not necessary after digital replantation. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, II.

FGF-2-containing dalteparin/protamine nanoparticles (FGF-2&D/P NPs) ameliorate UV-induced skin photoaging in hairless mice.

UVB exposure penetrates deeply into the dermis to alter skin barrier function, which is a primary factor in skin photoaging. We previously reported that dalteparin and protamine nanoparticles (D/P NPs) are effective carriers of FGF-2. This study aimed to examine the ability of FGF-2-containing D/P NPs (FGF-2&D/P NPs) to ameliorate UVB-induced skin photoaging in hairless mice. Dorsal skin of HR-1 hairless mice were exposed to UVB irradiation 5 days/week for 8 weeks (UV (+): final total, 2700 mJ/cm2). Mice were divided into four groups: Non-UVB (UV (-)) + saline, UV (+) + saline, UV (+) + FGF-2&D/P NPs, UV (+) + FGF-2, and UV (+) + D/P NPs, and following UVB irradiation, FGF-2&D/P NPs, FGF-2, and D/P NPs were applied to the groups of mice just after each UVB irradiation. Each group was subjected to evaluation of skin changes (elasticity), and histological examination using hematoxylin & eosin and terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) staining. UVB irradiation of mice significantly induced a decline in elasticity and acanthosis, which was alleviated by application of FGF-2&D/P NPs. Furthermore, TUNEL-staining showed the proportions of apoptotic dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) in the UV (+) + FGF-2&D/P NPs group were significantly lower than those in the UV (+) + saline, UV (+) + FGF-2, and UV (+) + D/P NPs groups. Thus, FGF-2&D/P NPs may be effective in preventing skin photoaging accelerated by UVB irradiation such as declining elasticity, acanthosis, and apoptosis of DFCs and EKCs.

Eyelid Reconstruction Using Oral Mucosa and Ear Cartilage Strips as Sandwich Grafting.

BACKGROUND: The eyelid structure can be divided into an inner layer and an outer layer. Reconstruction of a full-thickness eyelid defect is accomplished by full-thickness composite tissue transfer or combined layered reconstruction. We present a new technique for inner layer reconstruction using ear cartilage and oral mucosa. METHODS: The oral mucosa graft is harvested from the inner side of the lower lip to fit the defect size and shape. The ear cartilage graft is harvested as a rectangular strip. The harvested mucosa is sutured to the defect margin and the cartilage strip graft is interposed to the defect. Finally, the outer layer of the defect is covered with skin flaps. Consequently, the ear cartilage graft is sandwiched between the mucosa graft and the skin flap. RESULTS: We used this technique for the reconstruction of 13 full-thickness eyelid defects of various locations, sizes, and shapes. Ten cases involved the lower eyelid, 2 cases involved the lower eyelid including the medial canthus, and 1 case involved the upper eyelid. The oral mucosa graft survived in all patients. The reconstructions were successful and there were no postoperative reports of conjunctival or corneal irritation. CONCLUSIONS: The present technique using a combination of an ear cartilage strip graft and oral mucosa graft is an easy and versatile technique for reconstruction of inner layer eyelid defects. We believe that the beneficial effects of tears, which are richly oxygenated, improved survival of the grafted mucosa.

Maggot debridement therapy with a direct dressing can cause compression injuries in patients with chronic limb ischemia.

While there are no reports regarding dressing-associated iatrogenic skin ulcer as an adverse event of maggot debridement therapy (MDT), MDT is clinically used on patients with critical limb ischaemia with dermal fragility. Herein we report causes and counter measures for a case of iatrogenic skin ulcer induced by MDT dressing.

Maggot debridement therapy for a patient with critical limb ischaemia and severe cardiac dysfunction: possibility of limb salvage.

Ischaemic skin ulcer occurred on the foot of a 73-year-old man who had a history of fulminant myocarditis with severe cardiac dysfunction. We attempted wound bed preparation by maggot debridement therapy and salvaged his limb. It can be one of the adjuvant treatment strategies for critical limb ischaemia.

Coronary Artery Disease in Patients with Critical Limb Ischemia Undergoing Major Amputation or Not.

BACKGROUND: Due to the increase of elderly and diabetes patients, surgeons encounter patients requiring treatment of critical limb ischemia (CLI) in the presence of systemic arteriosclerotic diseases. In this study, we retrospectively investigated the prevalence of coronary artery disease (CAD) in patients with CLI who underwent major (above-the-ankle) amputation or nonmajor amputation (below-the-ankle amputation or debridement of wound). METHODS: We retrospectively investigated 129 consecutive patients surgically managed for CLI in our institution between January 2013 and December 2015. The prevalence of CAD was defined as a cardiac treatment history or significant vascular stenosis (stenosis of > 75%). The outcomes were compared between patients who underwent major amputation (n = 36) and nonmajor amputation (n = 93). Additionally, archived record of 566 patients treated nonsurgically by percutaneous transluminal angioplasty in our institution was investigated to evaluate patients with milder peripheral artery disease. RESULTS: CAD was present in 83 patients (69%), including 82% of patients who underwent major amputation and 63% of nonmajor amputation group. The prevalence of CAD was significantly higher in the major amputation group (P = 0.042). Ejection fraction was not significantly different (P > 0.05). Among the 566 CLI patients treated by only percutaneous transluminal angioplasty, 227 (40%) had CAD, which was a significantly lower prevalence than those surgically treated (P < 0.001). CONCLUSIONS: The presence of CAD is more frequent in CLI patients who require extended surgical management of the limb than in those who do not. Evaluation of CAD and careful perioperative management are important for patients with CLI patients.

The Effect of Smoking on Necrosis Rate in Digital Replantation and Revascularization with Prostaglandin E1 Therapy: A Retrospective Study.

BACKGROUND: Most microsurgeons believe that smoking and severity of injury adversely affect the outcome of digital replantation surgery. As countermeasures, several pharmacologic agents have been used for the perioperative period. The purpose of this retrospective study was to examine whether the rate of necrosis is appreciably different across smokers versus nonsmokers with prostaglandin E1 therapy. METHODS: The authors' study subjects included 144 patients (184 digits) who underwent replantation or revascularization between August of 2013 and August of 2015.The primary outcome was the incidence of total necrosis after replantation surgery, and the secondary outcomes were the rate of overall necrosis, proportion of total necrosis to overall necrosis, and total success. Intravenous administration of prostaglandin E1 was performed at the rate of 120 µg/day for 7 days after surgery in all patients. These outcomes of each injury type were compared between smoking and nonsmoking groups. RESULTS: Among the 184 injured digits, the incidence of total necrosis in smokers (23 percent) was higher than that in nonsmokers (17 percent), although no significant difference was shown (p = 0.36). The adjusted odds ratio was 1.17 (95 percent CI, 0.51 to 2.69). Similarly, there was no significant difference in the secondary outcomes between the two groups. CONCLUSION: The authors' retrospective study found no significant difference in the formation or extent of necrosis after replantation or revascularization between smoking and nonsmoking groups when all patients were treated with prostaglandin E1. CLINICAL QUESTION/LEVEL OF EVIDENCE: Risk, II.

Enhanced healing of mitomycin C-treated healing-impaired wounds in rats with PRP-containing fragmin/protamine microparticles (PRP&F/P MPs).

PURPOSE: The purpose of this study was to evaluate the accelerating effects of platelet-rich plasma-containing (PRP&) fragmin/protamine microparticles (F/P MPs) for repairing mitomycin C-treated healing-impaired wounds. Staining with terminal deoxynucleotidyl transferase-mediated dUTP nick-end labelling (TUNEL-staining) showed that apoptosis of dermal fibroblast cells (DFCs) and epidermal keratinocyte cells (EKCs) were significantly induced in the skin of the mitomycin C-treated rats. METHOD: Full-thickness skin defects were made on the back of rats and mitomycin C was applied on the wounds to prepare a healing-impaired wound. After washing out the mitomycin C, saline (control), F/P MPs alone, PRP alone, and PRP&F/P MPs were injected around the wounds. The rats were later euthanised and histological sections of the wounds were then prepared at indicated time periods after the treatment. RESULTS AND CONCLUSION: These results indicated the numbers of large, medium, and small capillary lumens 7 days after injection of PRP&F/P MPs were significantly higher than those after injection of PRP or F/P MPs alone. Furthermore, epithelium and granulation tissue formations were significantly stimulated in the healing-impaired wounds treated with PRP&F/P MPs 3, 7 and 14 days after injection of PRP&F/P MPs.

Three-dimensional culture using human plasma-medium gel with fragmin/protamine microparticles for proliferation of various human cells.

Fragmin/protamine microparticles (F/P MPs) have been used as carriers for the preservation and activation of cytokines in human plasma (HP)-Dulbecco's modified Eagle's medium (DMEM) gels. This study investigated a three-dimensional (3D) culture system using an HP-DMEM gel with 0.1 mg/mL F/P MPs and 5 ng/mL FGF-2 for the proliferation of human dermal fibroblast cells (DFCs), human microvascular endothelial cells (MVECs) and human coronary smooth muscle cells (SMCs), or 5 ng/mL interleukin (IL)-3/granulocyte-macrophage colony-stimulating factor (GM-CSF) for a human hematopoietic cell line (TF-1 cells). DFCs, MVECs, SMCs and TF-1 cells grew rapidly under 3D culture conditions using a low-concentration HP (2 %)-DMEM gel with F/P MPs and FGF-2 (for DFCs, MVECs and SMCs) or IL-3/GM-CSF (for TF-1 cells) at doubling times of 22, 23, 25 and 18 h, respectively, without the use of animal serum, compared to under 2D culture conditions using low-concentration human serum (2 %)-DMEM with 5 ng/mL FGF-2 or IL-3/GM-CSF on F/P MP-coated plates at doubling times of approximately 26, 25, 40 and 20 h, respectively.

Effective expansion of human adipose-derived stromal cells and bone marrow-derived mesenchymal stem cells cultured on a fragmin/protamine nanoparticles-coated substratum with human platelet-rich plasma.

Fragmin/protamine nanoparticles (F/P NPs) can be stably coated onto plastic surfaces and used as a substratum for the absorption and controlled release of growth factors (GFs) secreted from human platelet-rich plasma (PRP). In this study, we investigated the capability of F/P NP-coated plates to act as a substratum for the proliferation of human adipose-derived stromal cells (ASCs) and bone marrow-derived mesenchymal stem cells (BMSCs) with GFs in PRP. Both cell types adhered well to the F/P NP-coated plates and grew optimally, with a doubling time of 30 and 32 h in low-concentration PRP (0.5%) medium supplemented with 5 ng/ml fibroblast growth factor-2 (FGF-2) on the F/P NP-coated plates. These cells maintained their multilineage potential for differentiation into adipocytes or osteoblasts. Furthermore, ASCs and BMSCs grew well in medium without PRP and FGF-2 on F/P NP-coated plates pretreated with PRP and FGF-2 in a concentration-dependent manner. Thus, F/P NP-coated plates are a useful substratum for the adherence and proliferation of ASCs and BMSCs in low-concentration PRP medium supplemented with FGF-2. No xenogeneic serum is required.

Transplantation of inbred adipose-derived stromal cells in rats with plasma gel containing fragmin/protamine microparticles and FGF-2.

Fragmin/protamine microparticles (F/P MPs) have been used as a cell carrier for adipose-derived stromal cells (IR-ASCs) in inbred male Fisher 344 rats, and for preservation and controlled-release of fibroblast growth factor (FGF)-2 and various cytokines in inbred rat plasma (IRP)-DMEM (Dulbecco's modified Eagle's medium) gel. In this study, we investigated the capability of an IRP-DMEM gel containing F/P MPs and/or FGF-2, as a three-dimensional (3D)-culture, to expand IR-ASCs. We found that IR-ASCs grow faster under 3D-culture conditions in low IRP (3%)-DMEM gel containing F/P MPs and FGF-2 without any animal serum than those under 2D-culture in low inbred rat serum (3%)-DMEM with F/P MPs and FGF-2. About 0.3 mL of IR-ASCs (about 4,000,000 cells mL⁻¹) grown in IRP (6%)-DMEM gel containing F/P MPs and FGF-2 disappeared 8 days after subcutaneous injection in rats, suggesting that they are rapidly biodegradable. The number of large (diameter ≥200 µm or containing ≥100 erythrocytes), medium (diameter = 20-200 µm or containing 10-100 erythrocytes) and small (diameter ≤20 µm or containing 1-10 erythrocytes) capillaries after injection with IR-ASCs in an IRP-DMEM gel containing both F/P MPs and FGF-2, as well as the thickness of tissue granulation per microphotograph at the injected site, was significantly higher than those after injection with IR-ASCs in an IRP-DMEM gel containing either FGF-2 or F/P MPs. Thus, IRP-DMEM gel containing F/P MPs and FGF-2 are useful and safe IR-ASC carriers that facilitate cell proliferation, vascularization, and tissue granulation locally at injection sites.