One systemic administration of transforming growth factor-beta 1 reverses age- or glucocorticoid-impaired wound healing.

The role of intravenously administered recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) on the healing of incisional wounds in rats with impaired healing due to age or glucocorticoid administration was investigated. The administration of methylprednisolone to young adult rats decreased wound breaking strength to 50% of normal control. Breaking strength of incisional wounds from 19-mo-old rats was decreased approximately 27% compared with wounds from normal healing young adult rats. A single intravenous administration of rhTGF-beta 1 (100 or 500 micrograms/kg) increased wound breaking strength from old rats or young adult rats with glucocorticoid-induced impaired healing to levels similar to normal healing control animals when determined 7 d after injury. Even though the circulating half-life of systemically administered rhTGF-beta 1 is < 5 min, a sustained stimulatory effect on extracellular matrix secretion was evident in glucocorticoid-impaired rats when rhTGF-beta 1 was administered at the time of wounding, 4 h after wounding, or even 24 h before wounding. These observations indicate a previously unrecognized potential for the active form of TGF-beta 1 to profoundly influence the wound healing cascade after brief systemic exposure.

TGF-beta 1 induces bone closure of skull defects: temporal dynamics of bone formation in defects exposed to rhTGF-beta 1.

The temporal dynamics of bone repair in a skull defect in rabbits was examined to characterize the in vivo cellular events occurring following a single local application of recombinant human TGF-beta 1 (rhTGF-beta 1). Rabbits received vehicle or 0.4, 1, 2, or 5 micrograms rhTGF-beta 1 applied to 12 mm defects at the time of surgery. The defect sites were subsequently evaluated by radiography and qualitative and quantitative histology at time points ranging from 1 to 180 days. Based on radiographic assessment, the defect area decreased rapidly in a dose-dependent manner through 35 days after surgery in the rhTGF-beta 1-treated groups. Minimal closure occurred in sites administered vehicle control at all time points examined. Sites treated with rhTGF-beta 1 were characterized histologically by an increase in parameters of active bone formation through 49 days, including percentage osteoid surface, percentage osteoblast/total surface, and an increase in the trabecular bone volume. Bone resorption parameters were increased at 16 and 49 days with histologic evidence of remodeling from woven to lamellar bone. By 70 days, no differences were observed among the groups for parameters of either bone formation or resorption. Bone formation rate was not altered with rhTGF-beta 1 treatment at any time point. These results indicate that exogenously applied rhTGF-beta 1 stimulated the recruitment and proliferation of osteoblasts at the defect site, resulting in a rapid deposition of bony matrix, with normal remodeling processes occurring thereafter. This study supports the hypothesis that TGF-beta 1 is a potent osteoinductive growth factor in vivo and may have potential application as a therapeutic aid to nonhealing bony defects.

Rapid publication. TGF-beta 1 induces bone closure of skull defects.

Transforming growth factor beta 1 (TGF-beta 1) is a multifunctional regulatory protein. It is capable of inducing site-specific healing responses by increasing collagen synthesis and deposition as well as remodeling at sites of soft tissue repair. Large bony defects in the skull heal by fibrous connective tissue and never form bone unless osteoinductive bony fragments or powders are placed in the defect. We have found, however, that the single application of human recombinant TGF-beta 1 in a simple 3% methylcellulose gel to skull defects induced a dose-dependent increase in intramembranous bone formation. Complete bony bridging of defects occurred within 28 days after treatment with 2 micrograms TGF-beta 1. Sites treated with vehicle alone did not heal with bone formation but rather contained dense fibrous connective tissue between the defect margins.

In vivo induction of bone by recombinant human transforming growth factor beta 1.

A single application of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) adjacent to cartilage was found to induce bone formation in rabbit ear full-thickness skin wounds. At doses that optimally promote soft tissue healing, 25-100 ng rhTGF-beta 1 per wound caused osseous tissue formation starting 21 days after wounding to reach a peak incidence and area of bone formation at day 42. Bone formation was followed by active remodeling, resulting in lower incidence and area of bone formation at days 56 and 70. The early phase of bone formation was located overlying the cartilage and involved perichondrial cells that appeared to differentiate directly into osteoblasts forming bone matrix without a cartilage precursor. Cartilage was replaced with bone at later time points. rhTGF-beta 1 was able to increase the ratio of osteoblasts to osteoclasts lining the trabecular surface of bone and thus increase the net amount of bone formation. The present studies suggest a potential therapeutic role for rhTGF-beta 1 in hard tissue repair.

Characterization of radioiodinated recombinant human TGF-beta 1 binding to bone matrix within rabbit skull defects.

Bone healing is regulated in part by the local production of TGF-beta 1 and other growth factors produced by cells at the site of injury. The single application of recombinant human TGF-beta 1 (rhTGF-beta 1) to calvarial defects in rabbits induces an accelerated recruitment and proliferation of osteoblasts within 3 days. This ultimately results in the formation of new bone and the complete closure of the defect within 28 days. The persistence and localization of [125I]rhTGF-beta 1 within an osseous defect was investigated after applying a single dose of [125I]rhTGF-beta 1 formulated in a 3% methylcellulose vehicle. Normal bone encompassing the defect site, the periosteum, and the gel film covering the dura were harvested at 0, 4, 8, and 24 h and 3, 7, and 16 days after [125I]rhTGF-beta 1 application. The defect site-associated radioactivity was quantitated, visualized by autoradiography, and characterized by TCA precipitation and SDS-PAGE. Radioactivity was observed in autoradiographs of gross specimens, histologic sections of the bone matrix, and periosteal tissue surrounding the defect. There was a time-dependent decrease in TCA-precipitable radioactivity; however, radioactivity was still associated with the bone matrix 16 days after application of [125I]rhTGF-beta 1. SDS-PAGE and autoradiography of the radioactivity in homogenized bone and periosteal samples revealed a 25 kD band, suggesting that the radioactivity remaining at the defect site represented intact [125I]rhTGF-beta 1.(ABSTRACT TRUNCATED AT 250 WORDS)

Human recombinant transforming growth factor-beta 1 modulation of biochemical and cellular events in healing of ulcer wounds.

The effects of recombinant human transforming growth factor beta 1 (rhTGF-beta 1) on wound healing were examined in a rabbit ear ulcer model in which rhTGF-beta 1 was applied to full-thickness biopsy ulcers on the ears. The influence of perichondrium on healing was studied by comparing ulcers with and without perichondrium on 1) formation of total healing wound area (HWA, the newly formed connective and granulation tissues within the ulcer) over time and 2) the amount of collagen synthesized by the wound tissue at day 5. The HWA of ulcers with intact perichondrium increased sharply with time and reached a plateau at day 7, whereas a slower healing occurred in the perichondrium-free model where maximal HWA appeared at day 14. Topical application of 100 ng of rhTGF-beta 1 per wound accelerated healing by increasing HWA in both models. The enhancement of healing by rhTGF-beta 1 was associated with increased collagen synthesis. The percent collagen synthesis in the rhTGF-beta 1 was doubled in the perichondrium-intact ulcers and increased 40% in the perichondrium-free ulcers. DNA synthesis in the perichondrium-intact ulcers was not altered by rhTGF-beta 1 when measured at day 5 by in vitro labeling with [3H]thymidine ([3H]TdR). Autoradiography indicated that the primary cells labeled in the wound tissue were epithelial cells and rhTGF-beta 1 enhanced the migration of these cells from the wound margin towards the center. To evaluate the effects of rhTGF-beta 1 on fibroblasts derived from the granulation tissue of the wound, cells were treated with increasing concentrations of rhTGF-beta 1 and DNA and collagen synthesis were determined. rhTGF-beta 1 elicited a biphasic change in percent collagen synthesis with a maximal increase of 50% at 20 pM followed by a decline. A twofold increase in [3H]TdR incorporation that plateaued at 1 nM was also observed. Our results indicate that the cellular responses to rhTGF-beta 1 differ in vivo and in vitro. The perichondrium-intact ulcers contain more wound tissue and have larger responses to rhTGF-beta 1 stimulation, which allows better examination of biochemical and cellular events. The in vivo mechanisms are multi-factorial, which may involve cell migration and recruitment as results of numerous cell/cell and cell/matrix interactions.

Human relaxin decreases collagen accumulation in vivo in two rodent models of fibrosis.

The reproductive hormone, relaxin, inhibits collagen synthesis in vitro by normal human dermal fibroblast. In the present study, recombinant human relaxin is shown to modulate collagen accumulation and organization by mesenchymal cells in vivo in two rodent models of fibrosis: 1) fibrotic infiltration of polyvinyl alcohol sponge implants in rats, and 2) capsule formation around implanted osmotic pumps in mice. In the sponge, relaxin inhibits collagen accumulation, a measured by hydroxyproline content, in a dose-responsive manner by up to 25-29% in animals receiving 30 ng/ml relaxin, a finding supported by a decrease in collagen-specific trichrome staining in sections of sponges from relaxin-treated animals. In mice, the capsules surrounding relaxin-containing pumps are thinner and less dense than are capsules from control pumps. Ultrastructurally, control capsules are composed of densely packed parallel arrays of collagen fibrils, whereas fibrils more frequently are not packed in parallel arrays and are less abundant in treated capsules.

Transforming growth factor-beta. Effect on soft tissue repair.

Previous studies have demonstrated that TGF-beta possesses many of the biologic properties necessary for acceleration of the normal wound healing process. We report that recombinant human TGF-beta 2 (rhuTGF-beta 1) increases wound strength and accelerates wound closure when applied topically to experimental wounds. Doses of 5 to 1,000 ng/wound increased wound strength in a dose-response manner and wound strength increase as high as 161% above control in the rat incisional wound model. Increased wound strength was observed as early as 3 days following rhuTGF-beta 1 application and continued to Day 28. In the rabbit ear ulcer model, acceleration of wound closure was observed following doses of 5 to 100 ng/wound applied a single topical application. No adverse effects of rhuTGF-beta 1 were observed. The amount of fibrous tissue, scar formation, and mitotic figures were not significantly greater than control. Epithelialization of rhuTGF-beta 1-treated wounds was not impeded. rhuTGF-beta 1 induced bone formation in the rabbit ear ulcer model but not in the rat incisional model, suggesting that precursor cells, such as perichondrial cells, are required for the bone forming activities of TGF-beta 1.

An intraosseous device for studies of bone-healing. The effect of transforming growth-factor beta.

A novel implantable device, the analytic bone implant, was used in order to establish a model for studies of bone-healing and the evaluation of factors that augment the process, such as transforming growth-factor beta (TGF-beta). This device was implanted into the tibiae of four baboons. After healing, bone was removed from the center chamber. Recombinant human TGF beta-1 was then delivered to the core of the device. After twenty-two days of healing, the device was disassembled and the newly formed bone was removed from the core of the implant for histomorphometric analysis. An analysis of the bone revealed a substantial effect of TGF-beta on osteoblastic activity and proliferation compared with that seen in control and placebo groups. However, despite increased osteoblastic activity, trabecular bone volumes at twenty-two days were equivalent among the groups. The number of osteoclasts and the erosion of the surface were also increased, although not significantly so. Substantial endochondral formation of bone was seen in the supraperiosteal tissues directly over the implants that contained TGF-beta but not over the implants in the control and placebo groups. These data demonstrate the utility of this bone-implant model for studies of bone-healing with minimally invasive methods. In addition, use of the device provided the first in vivo data on the effects of TGF-beta at an intermediate (twenty-two-day) time-point in the healing process in a non-human primate.

Combination of bone marrow and TGF-beta1 augment the healing of critical-sized bone defects.

A 1.5 cm segmental defect in the radius of rabbits was used to compare healing at sites administered TGF-beta, with or without autologous bone marrow, to autogenous cortical bone graft. The carrier for TGF-beta consisted of tricalcium phosphate (TCP) granules and hetastarch. The efficacy of TGF-beta formulations and bone marrow (BM) was compared to autogenous bone, carrier control, and untreated defect sites. Bone measurements taken at necropsy included the anterior-posterior (AP) diameter and medial to lateral (LAT) diameter of the defect; the AP and LAT diameters of both radii measured 1 cm proximal to the distal epiphysis, and the AP and LAT diameters of the mid-shaft of the femora. The bones from each group were subdivided for either histological evaluation or for mechanical testing. Strength (maximum torque), energy, angle of rotation and stiffness were determined for both the treated and contralateral radii. Results of the radiographic, necropsy, and mechanical data for defects administered 1.0 microgram of TGF-beta1 + BM or autogenous cortical bone were similar and indicated superior healing compared to defects left blank or administered the carrier control with or without bone marrow. Defects administered 1.0 microgram of TGF-beta1 + BM or autogenous cortical bone had high mechanical strength relative to the control groups and were characterized histologically as healed primarily with lamellar bone. The results from the defects left blank or administered carrier control were similar and generally characterized by poor healing or nonunion. This study demonstrated substantial equality of healing between 1.0 microgram of TGF-beta1 + BM and autograft indicating that this formulation could function as a substitute for autologous grafts.

TGF-beta and wound healing.

Wound healing comprises an ordered sequence of events including cell migration and proliferation, synthesis of extracellular matrix, angiogenesis and remodelling. TGF-beta regulates many of these processes. Animal models are used to study healing of simple linear incision wounds and deeper dermal wounds under normal and impaired conditions. TGF-beta increases the rate of healing and the breaking strength of the repaired tissue. It also enhances angiogenesis and consequent blood flow to dermal wounds, partly by stimulating the local release of other growth factors. TGF-beta reverses the adverse affects of glucocorticoids on wound healing and thus may be useful in the treatment of chronic ulcers or wounds in patients whose normal responses have been impaired by therapy with steroids, radiation or other drugs.

Regulation of experimental autoimmune neuritis by transforming growth factor-beta 1.

Experimental autoimmune neuritis (EAN) is a T-cell-mediated autoimmune disease characterized by demyelination and mononuclear cell infiltration of the peripheral nervous system. It is induced in Lewis rats by administration of myelin P2 protein or a synthetic peptide (SP-26) corresponding to amino acid residues 53-78 of bovine P2 protein. The effects of transforming growth factor-beta 1 (TGF-beta 1) on the clinical signs, histological changes, cell-mediated immune responses, and secretion of interferon-gamma (IFN-gamma) by lymphoid cells of rats with EAN were examined. Systemic administration of TGF-beta 1 markedly inhibited the clinical signs and histological changes of EAN when given intraperitoneally every other day for Days 0 through 18. In addition, it decreased proliferative responses and reduced the delayed-type hypersensitivity (DTH) response to SP-26 compared to control rats. The reduction in clinical severity correlated with skin test unresponsiveness (DTH) to the disease-inducing agent (SP-26) as well to decreased cellular responsiveness to the antigen in vitro. The decrease in cellular responsiveness extended to a decrease in at least one T cell lymphokine, IFN-gamma. The profound effect of TGF-beta on disease progression in EAN, a T-cell-mediated process, is consistent with a direct effect of this growth factor on T lymphocytes.

Crohn's disease of the vulva.

Vulvar involvement is an uncommon extraintestinal manifestation of Crohn's disease. A case of "metastatic" vulvar Crohn's disease occurring in a 23-yr-old woman, status post-ileocolectomy, is described. The diagnosis was established by vulvar punch biopsy, and a therapeutic response was achieved with metronidazole and prednisone. The literature on vulvar involvement with Crohn's disease is reviewed and therapeutic options are discussed.

Recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) enhances healing and strength of granulation skin wounds.

A new animal model to study secondary intention wound healing and the effects of topically applied rhTGF-beta 1 was developed. A time course study was performed of full thickness 6 mm punch wounds placed on the backs of anesthetized pigs and treated once with either 3% methylcellulose or rhTGF-beta 1 in 3% methylcellulose or left untreated. Wounds receiving rhTGF-beta 1 had enhanced tensile strength at days 4 and 7 compared to controls. Studies of the response on days 4 and 7 to graded doses of rhTGF-beta 1 showed that a dose of 250 or 2500 ng rhTGF-beta 1 gave a similar enhanced wound strength, while 25 ng rhTGF-beta 1 had no effect. Blood flow to treated granulating wounds as measured by 141Ce microspheres indicate an increase in flow in wounds treated with 250, 500 or 2500 ng rhTGF-beta 1 compared to controls. These results indicate a possible use for rhTGF-beta 1 in enhancing wound healing clinically.

Treatment of left-sided ulcerative colitis with 4-aminosalicylic acid enemas. A double-blind, placebo-controlled trial.

Twenty-five patients with active left-sided ulcerative colitis were randomly assigned to receive either 2 g of 4-aminosalicylic acid (para-aminosalicylic acid) or placebo in a 60-mL volume as a nightly retention enema. The duration of treatment was 8 weeks. Disease activity was assessed by grading clinical symptoms of blood, mucus, urgency, sigmoidoscopic findings, and degree of histologic inflammation in rectal biopsies. At 8 weeks, 10 of 12 patients (83%; 95% confidence interval [CI], 55% to 97%) who received 4-aminosalicylic acid showed improvement in clinical, sigmoidoscopic, and histologic variables. In contrast, only 2 of 13 patients (15%, 95% CI, 4% to 38%) who had received placebo showed clinical improvement (P less than 0.005). The 11 patients in the placebo group who showed no improvement were treated subsequently with open-label 4-aminosalicylic acid enemas. Of the 11, 9 showed clinical, sigmoidoscopic, and histologic improvement. No adverse effects were seen. 4-Aminosalicylic acid enemas are a safe and effective means of treating left-sided ulcerative colitis.

Effects of growth factors and L-arginine on ischemic skin flaps in rats.

This study determined the effect of the polypeptide growth factors transforming growth factor-beta (TGF-beta), insulin-like growth factor-I (IGF-I), and growth hormone (GH) alone and in combination with dietary L-Arginine HCL (ARG) on skin flap survival in rats. Caudally based dorsal skin flaps were created in 110 Sprague-Dawley rats. The rats were randomly assigned into three treatment groups, based on drinking water supplementation. Group 1 (n = 50) received ARG in their drinking water, group 2 (n = 50) received tap water alone, and group 3 (n = 10) received N-omega-nitro-L-arginine (L-NA) and hydralazine. Groups 1 and 2 were divided into subgroups of 10 rats each based on treatment with either: TGF-beta, IGF-I, GH, or IGF-I + GH. All subgroups that received GH had significantly greater (P < .0001) median body weight gains when compared with subgroups not receiving GH. L-arginine HCL when added to IGF-I negated the positive effects of IGF-I on both flap survival and weight gain. Although the rats in all subgroups from groups 1 and 2 had an increase in mean percent skin flap survival when compared with the water alone subgroup, only rats receiving IGF-I, or the combination of ARG with either TGF-beta or GH, had statistically significant enhanced skin flap survival. Rats in group 3 did not show an increase in skin flap survival when compared with the control subgroup.

TGF-beta 1 accelerates wound healing: reversal of steroid-impaired healing in rats and rabbits.

TGF-beta modulates events of normal wound healing through multiple pathways that influence cell infiltration, proliferation, angiogenesis, extracellular matrix synthesis and remodeling. The effects of topically applied TGF-beta 1 on wound healing in two models of healing were evaluated when the healing response was impaired by the administration of methylprednisolone to rats or rabbits. TGF-beta 1 increased the healing of linear incision wounds on rats, as measured by breaking strength, to that of normal rats. Full thickness open wounds were also created on the inner ears of rabbits to simulate a non-contracting wound with limited blood supply. Healing was further impaired by the administration of methylprednisolone. The single application of TGF-beta 1 improved the healing of open wounds. TGF-beta 1 stimulated increased granulation tissue formation, as well as reepithelialization. The amount of granulation tissue and epithelialization were similar to wounds from normal-healing control rabbits. The delayed healing caused by methylprednisolone permitted the evaluation of multiple applications of TGF-beta 1 to wounds. Two applications of TGF-beta 1 spaced 7 days apart further improved the healing response when compared to a single application. Thus, single or multiple topical applications of TGF-beta 1 reversed impaired healing conditions secondary to methylprednisolone when used on incisional or open wounds. These observations support the hypothesis that growth factors, such as TGF-beta 1, may be useful as accelerators of wound repair in patients with impaired healing conditions.

Development of tricalcium phosphate/amylopectin paste combined with recombinant human transforming growth factor beta 1 as a bone defect filler.

Tricalcium phosphate (TCP) was combined with amylopectin to form a deliverable carrier paste for recombinant human transforming growth factor beta 1 (rhTGF-beta 1) intended for bone repair applications. Approximately 80% of rhTGF-beta 1 was released from the carrier within 24 h following in vitro incubation in serum. Full biological activity was maintained, suggesting the growth factor was stable in this formulation before and after in vitro release. In vivo efficacy also was assessed, in comparison to a sham control group and a placebo-treated group, using a rabbit unilateral segmental defect model (1 cm). Radiographs of defect sites taken at scheduled intervals and the mechanical testing of treated limbs at 56 days demonstrated a higher incidence of radiographic bone union, in concert with a stronger torque strength, in the rhTGF-beta 1-treated group compared to the placebo group. The short duration of the study and the fact that the model used was not a critical defect may account for the lack of superiority of the rhTGF-beta 1-treated group over the healing of the sham control. The in vivo pharmacokinetics of the growth factor evaluated in the same rabbit model suggested that rhTGF-beta 1 persisted intact at the defect site for more than 21 days. Gamma imaging and radioactivity recovery at defects administered to [131I]- and [125I]-labeled rhTGF-beta 1, respectively, estimated the half-life of rhTGF-beta 1 eliminated from the applied site to be 4-6 days. The present report substantiates the potential of rhTGF-beta 1 and its carrier for treatment of bone defects.

Accelerated healing of ulcer wounds in the rabbit ear by recombinant human transforming growth factor-beta 1.

A dermal ulcer wound-healing model was established in rabbit ear to examine the effects of recombinant human transforming growth factor-beta 1 (rhTGF-beta 1) in wound healing. Histomorphometric examination of the wounds indicate a biphasic healing response 7 days after a single application of rhTGF-beta 1 at the time of wounding. Statistically significant healing occurred at 5-100 ng but not at higher doses of 500 or 1000 ng rhTGF-beta 1/wound. Enhanced collagen synthesis as determined by [3H]proline incorporation occurred at 15 and 25 ng and was significantly depressed at 500 ng rhTGF-beta 1/wound. Multiple doses of 100 ng rhTGF-beta 1 applied to the wound at the time of wounding and for 3 days after wounding provided results comparable to the single application of growth factor. Delaying treatment 24 hr after wounding did not enhance wound healing compared with vehicle. Our findings suggest that rhTGF-beta 1 can be a valuable growth factor to improve the healing of ulcer wounds.