Efficient autocrine and paracrine signaling explain the osteogenic superiority of transgenic BMP-2 over rhBMP-2.

Bone morphogenetic protein-2 (BMP-2) is an osteogenic protein used clinically to enhance bone healing. However, it must be applied in very high doses, causing adverse side effects and increasing costs while providing only incremental benefit. Preclinical models of bone healing using gene transfer to deliver BMP-2 suggest that transgenic BMP-2 is much more osteogenic than rhBMP-2. Using a reporter mesenchymal cell line, we found transgenic human BMP-2 cDNA to be at least 100-fold more effective than rhBMP-2 in signaling. Moreover, a substantial portion of the BMP-2 produced by the transduced cells remained cell associated. Signaling by transgenic BMP-2 occurred via binding to the type I receptor, activating the associated kinase and generating phospho-smads. Signaling was partially resistant to noggin, an important extracellular inhibitor of BMP-2, possibly because nascent BMP-2 binds to its cell surface receptor during secretion and thus signals in a protected peri-cellular environment. Although the amounts of BMP-2 secreted by the transduced cells were too low to affect distant cells, transduced cells were able to induce signaling in a paracrine fashion that required close proximity of the cells, possibly cell-to-cell contact. The greater osteogenic potency of transgenic BMP-2 was confirmed with human bone marrow stromal cells.

Segmental defect healing in the presence or absence of recombinant human BMP2: Novel insights from a rat model.

This study defined and compared the course of native, impaired and growth factor-stimulated bone regeneration in a rat femoral defect model. A mid-diaphyseal defect with rigid internal fixation was surgically created in the right femur of male Fischer rats and serially analyzed over 36 weeks. Native bone regeneration was modeled using a sub-critical, 1 mm size defect, which healed uneventfully. Critical size defects of 5 mm were used to analyze impaired bone regeneration. In a third group, the 5 mm defects were filled with 11 µg of recombinant human bone morphogenetic protein 2 (rhBMP2) impregnated onto an absorbable collagen sponge, modeling its clinical use. Native bone regeneration was characterized by endochondral ossification with progressive remodeling to ultimately resemble intact femora. An endochondral response was also observed under conditions of impaired bone regeneration, but by week 8 medullary capping occurred with fibrofatty consolidation of the tissue within the defect, resembling an atrophic non-union. rhBMP2 treatment was associated with prolonged inflammatory cytokine expression and rapid intramembranous bone formation occurring with reduced expression of cartilage-associated collagens. Between weeks 4 and 36, rhBMP2-treated bones demonstrated decreased trabecular number and increased trabecular separation, which resulted in inferior mechanical properties compared with bones that healed naturally. Clinical Significance: Recombinant human bone morphogenetic protein 2 (rhBMP2) is used clinically to promote healing of long bones. Our data suggest that it drives intramembraneous ossification producing an inferior regenerate that deteriorates with time. Clinical outcomes would be improved by technologies favoring endochondral regenerative ossification.

Prevalence of AAV2.5 neutralizing antibodies in synovial fluid and serum of patients with osteoarthritis.

Osteoarthritis (OA) is a leading cause of disability with no cure and only supportive therapy. Adeno-associated virus (AAV) serotype 2.5 is being used in a Phase I clinical trial to deliver the interleukin-1 receptor antagonist into knee joints with OA. Neutralizing antibodies (Nab) directed against AAV2.5, if present, could inhibit gene transfer. Here, we report the prevalence of AAV2.5 Nab in the sera and synovial fluids of patients with OA. Nab titers were measured by their ability to inhibit in vitro transduction by AAV2.5 encoding GFP. Of 44 synovial fluids from patients with mid-stage and advanced OA, 43% had undetectable Nab; 25% had low titers (<1:100), 16% had medium titers (1:100-1:1000) and 16% had high titers (>1:1000) of Nab. Titers of AAV2.5 Nabs correlated with those of AAV2, but not with those of AAV5. Serum titers of AAV2.5 Nab correlated positively with titers in synovial fluid, and were never less than the matched synovial fluid titers. These findings suggest that high titers of Nab against AAV2.5 are uncommon in the synovial fluids of patients with OA, and individuals with high synovial fluid Nab titers can be identified by measuring titers in the serum.

IL-1Ra gene transfer potentiates BMP2-mediated bone healing by redirecting osteogenesis toward endochondral ossification.

An estimated 100,000 patients each year in the United States suffer severe disability from bone defects that fail to heal, a condition where bone-regenerative therapies could provide substantial clinical benefits. Although recombinant human bone morphogenetic protein-2 (rhBMP2) is an osteogenic growth factor that is clinically approved for this purpose, it is only effective when used at exceedingly high doses that incur substantial costs, induce severe inflammation, produce adverse side effects, and form morphologically abnormal bone. Using a validated rat femoral segmental defect model, we show that bone formed in response to clinically relevant doses of rhBMP2 is accompanied by elevated expression of interleukin-1 (IL-1). Local delivery of cDNA encoding the IL-1 receptor antagonist (IL-1Ra) achieved bridging of segmental, critical size defects in bone with a 90% lower dose of rhBMP2. Unlike use of high-dose rhBMP2, bone formation in the presence of IL-1Ra occurred via the native process of endochondral ossification, resulting in improved quality without sacrificing the mechanical properties of the regenerated bone. Our results demonstrate that local immunomodulation may permit effective use of growth factors at lower doses to recapitulate more precisely the native biology of healing, leading to higher-quality tissue regeneration.

Enthesis Healing Is Dependent on Scaffold Interphase Morphology-Results from a Rodent Patellar Model.

The use of multiphasic scaffolds to treat injured tendon-to-bone entheses has shown promising results in vitro. Here, we used two versions of a biphasic silk fibroin scaffold to treat an enthesis defect created in a rat patellar model in vivo. One version presented a mixed transition between the bony and the tendon end of the construct (S-MT) while this transition was abrupt in the second version (S-AT). At 12 weeks after surgery, the S-MT scaffold promoted better healing of the injured enthesis, with minimal undesired ossification of the insertion area. The expression of tenogenic and chondrogenic markers was sustained for longer in the S-MT-treated group and the tangent modulus of the S-MT-treated samples was similar to the native tissue at 12 weeks while that of the S-AT-treated enthesis was lower. Our study highlights the important role of the transition zone of multiphasic scaffolds in the treatment of complex interphase tissues such as the tendon-to-bone enthesis.

Efficient healing of large osseous segmental defects using optimized chemically modified messenger RNA encoding BMP-2.

Large segmental osseous defects heal poorly. Recombinant, human bone morphogenetic protein-2 (rhBMP-2) is used clinically to promote bone healing, but it is applied at very high doses that cause adverse side effects and raise costs while providing only incremental benefit. We describe a previously unexplored, alternative approach to bone regeneration using chemically modified messenger RNA (cmRNA). An optimized cmRNA encoding BMP-2 was delivered to critical-sized femoral osteotomies in rats. The cmRNA remained orthotopically localized and generated BMP locally for several days. Defects healed at doses ≥25 µg of BMP-2 cmRNA. By 4 weeks, all animals treated with 50 µg of BMP-2 cmRNA had bridged bone defects without forming the massive callus seen with rhBMP-2. Moreover, such defects recovered normal mechanical strength quicker and initiated bone remodeling faster. cmRNA regenerated bone via endochondral ossification, whereas rhBMP-2 drove intramembranous osteogenesis; cmRNA provides an innovative, safe, and highly translatable technology for bone healing.

Picrosirius Red Staining: Revisiting Its Application to the Qualitative and Quantitative Assessment of Collagen Type I and Type III in Tendon.

Collagen has a major role in the structural organization of tendons. Picrosirius red (PSR) staining viewed under polarized light microscopy is the standard method to evaluate the organization of collagen fibers in tissues. It is also used to distinguish between type I and type III collagen in tissue sections. However, accurate analysis and interpretation of PSR images are challenging because of technical factors and historical misconceptions. The aim of this study was to clarify whether collagen types I and III can be distinguished by PSR staining in rat Achilles tendons, using double immunohistochemistry as the positive control. Our findings showed that PSR staining viewed with polarized light microscopy was suitable for qualitative and quantitative assessment of total collagen but was not able to distinguish collagen types. We found it critical to use a polarizing microscope equipped with a rotating stage; tendon section orientation at 45° with respect to crossed polarizers was optimal for the qualitative and quantitative assessment of collagen organization. Immunohistochemistry was superior to PSR staining for detection of collagen type III. We also compared formalin and Bouin solution as fixatives. Both produced similar birefringence, but formalin-fixed tendons provided higher quality histological detail with both hematoxylin-eosin and immunostaining.

An Improved, Chemically Modified RNA Encoding BMP-2 Enhances Osteogenesis In Vitro and In Vivo.

IMPACT STATEMENT: The use of chemically modified RNA (cmRNA) with increased stability using translation initiator of short untranslated regions (TISU) offers the prospect of finally allowing us to unlock the potent osteogenic properties of BMP-2 in a clinically expedient manner. As noted, delivery of recombinant BMP-2 protein has had modest clinical efficacy, whereas gene delivery is effective but very difficult to translate into human clinical use. This study shows the great potential of cmRNA encoding BMP-2 with TISU in a long-bone critical-sized rat model.

A stimulatory thyrotropin receptor antibody (M22) and thyrotropin increase interleukin-6 expression and secretion in Graves' orbital preadipocyte fibroblasts.

BACKGROUND: Patients with Graves' ophthalmopathy (GO) have circulating autoantibodies directed against the thyrotropin receptor (TSHR) and elevated levels of the proinflammatory cytokine interleukin-6 (IL-6) in both serum and orbital tissues. We hypothesized that these autoantibodies might increase IL-6 expression and secretion in preadipocyte fibroblasts and adipocytes from patients with GO, and thus directly impact the clinical activity of the disease. METHODS: IL-6 mRNA levels were measured in cultures of GO orbital preadipocytes (n = 3) treated during adipocyte differentiation with a monoclonal stimulatory TSHR antibody (M22; 10 ng/mL), IL-6 (1 ng/mL), or TSH (10 U/L). Additionally, levels of IL-6 protein secretion were assessed after adipocyte differentiation in orbital cultures exposed to TSH or M22 for 24 or 48 hours (n = 8). IL-6 mRNA levels were also measured in orbital adipose tissue specimens from well-characterized GO patients (n = 9) and normal individuals (n = 9). RESULTS: Treatment of GO orbital preadipocyte cultures with IL-6, TSH, or M22 during adipocyte differentiation resulted in increased IL-6 mRNA levels (3.1-fold, 2.9-fold, and 2.7-fold, respectively; p < 0.05). Treatment of orbital cultures with M22 or TSH after adipocyte differentiation enhanced the release of IL-6 protein into the medium at both 24 and 48 hours for TSH (mean 1.9- and 2.3-fold; p = 0.002 and 0.015, respectively) and at 48 hours for M22 (mean 2.0-fold; p = 0.005). In addition, we found mean IL-6 mRNA levels to be significantly increased in GO orbital adipose tissue specimens (10-fold; p < 0.01), primarily attributable to high levels in three of the four patients with clinical activity scores >or=5. CONCLUSIONS: Both TSH and M22 increase IL-6 expression in orbital preadipocyte fibroblasts and IL-6 secretion by mature adipocytes. These results suggest that circulating TSHR autoantibodies in GO might play a direct role in the clinical activity of the disease.

Eosinophil ribonucleases and their cutaneous lesion-forming activity.

Eosinophil granule proteins are deposited in cutaneous lesions in many human diseases, but how these proteins contribute to pathophysiology is obscure. We injected eosinophil cationic protein (ECP or RNase 3), eosinophil-derived neurotoxin (EDN or RNase 2), eosinophil peroxidase (EPO), and major basic protein-1 (MBP1) intradermally into guinea pig and rabbit skin. ECP and EDN each induced distinct skin lesions at >or=2.5 microM that began at 2 days, peaking at approximately 7 days and persisting up to 6 wk. These lesions were ulcerated (ECP) or crusted (EDN) with marked cellular infiltration. EPO and MBP1 (10 microM) each produced perceptible induration and erythema with moderate cellular infiltration resolving within 2 wk. ECP and EDN localized to dermal cells within 2 days, whereas EPO and MBP1 remained extracellular. Overall, cellular localization and RNase activity of ECP and EDN were critical for lesion formation; differential glycosylation, net cationic charge, or RNase activity alone did not account for lesion formation. Ulcerated lesions from patients with the hypereosinophilic syndrome showed ECP and EDN deposition comparable to that in guinea pig skin. In conclusion, ECP and EDN disrupt skin integrity and cause inflammation. Their presence in ulcerative skin lesions may explain certain findings in human eosinophil-associated diseases.

Evidence for enhanced Thy-1 (CD90) expression in orbital fibroblasts of patients with Graves' ophthalmopathy.

BACKGROUND: Thy-1 is a surface protein that defines functionally distinct subpopulations of fibroblasts, with those lacking the antigen being capable of adipogenesis. Because increased fat cell development is a hallmark of the orbit in Graves' ophthalmopathy (GO), we wished to compare baseline Thy-1 expression in orbital fibroblasts from GO patients and normal individuals, and determine whether levels of the protein might be impacted by adipogenesis following peroxisome proliferator activator-gamma ligation. METHODS: Orbital adipose/connective tissue specimens were obtained from euthyroid patients undergoing orbital decompression surgery for severe GO (n = 9) and from normal individuals (n = 9). Thy-1 mRNA and protein levels were assessed in tissue specimens and in orbital fibroblast cultures at baseline using RT-PCR, quantitative immunofluorescent staining, and flow cytometry using a specific Thy-1 mouse anti-human CD90/Thy-1 monoclonal antibody. In addition, some orbital fibroblast cultures were treated with rosiglitazone (1 microL/mL; 2 nM) or control for 10 days in culture. RESULTS: We found that Thy-1 mRNA and protein expression was higher in uncultured GO connective/adipose tissue specimens (3.8-fold; 0.835 +/- 0.116 relative expression) compared with normal (0.22 +/- 0.062; p = 0.002) and in cultured orbital fibroblasts from GO patients (3.3-fold; 9.28 +/- 1.82 relative expression) compared with normal cultures (2.80 +/- 0.42; p = 0.013). Adipocyte differentiation had no effect on Thy-1 expression. Flow cytometry and immunofluorescent staining showed increased numbers of Thy-1-positive cells in the GO (mean 77.9 + 4.09%; range 66.5-84.8%) compared with the normal fibroblast cultures (66.8 + 1.6%; range 63.3-71.0% positive; p = 0.046), as well as higher levels of expression on the positive cells. CONCLUSIONS: Increased Thy-1 expression in GO orbital tissues and cultures is likely a consequence of the orbital disease process, reflecting both the presence of increased numbers of Thy-1-positive cells and higher expression on those cells. Adipogenesis itself does not appear to impact Thy-1 expression. Increased expression of this protein in GO could represent an adaptive response to cell injury, in effect limiting disease progression within the orbital adipose/connective tissues.

Thyroid transcription factor-1 in orbital adipose tissues: potential role in orbital thyrotropin receptor expression.

Thyroid transcription factor-1 (TTF-1) is required for maximal expression of thyrotropin receptor (TSHR) in the thyroid. Extrathyroidal TSHR expression is detectable in normal orbital adipose tissues, with increased levels found in orbital tissues from patients with Graves' ophthalmopathy (GO), and in orbital preadipocyte cultures following differentiation. In order to determine whether TTF-1 might be involved in orbital TSHR expression, we used quantitative real-time polymerase chain reaction (PCR) to assess relative expression of this and other thyroid-associated transcription factors (TTF-2 and Pax-8) in GO orbital tissue specimens (n = 28) and cultures (n = 3), and in normal orbital tissues (n = 19) and cultures (n = 3). We detected TTF-1 and TTF-2 mRNA in GO and normal orbital tissue samples, with no difference in levels noted between the tissues. In the GO orbital cultures, TTF-1 mRNA was higher in differentiated than in control (undifferentiated) cultures (p < 0.05), while TTF-2 was unchanged. In the normal cultures, neither TTF-1 nor TTF-2 mRNA levels increased in differentiated cultures. Pax8 was undetectable in all orbital tissues and cell cultures. The presence of mRNA encoding TTF-1 in orbital tissues and cultures suggest that this transcription factor may play an important role in extrathyroidal, as it does in thyroidal, TSHR expression.

Gene expression profiling of orbital adipose tissue from patients with Graves' ophthalmopathy: a potential role for secreted frizzled-related protein-1 in orbital adipogenesis.

CONTEXT: The signs and symptoms of Graves' ophthalmopathy (GO) result from inflammation and increased volume of the orbital adipose tissues and extraocular muscles. OBJECTIVE: Our objective was to identify differentially regulated genes that may be involved in stimulating the orbital adipose tissue expansion seen in GO. DESIGN: Gene expression profiling was used to compare genes expressed in orbital adipose tissues from GO patients and normal individuals. SETTING: The study took place at a private practice tertiary referral center. PATIENTS: Orbital adipose tissues were collected at transantral orbital decompression surgery from 20 euthyroid patients undergoing this procedure for severe GO and at early autopsy from eight normal individuals having no evidence of thyroid or ocular disease. RESULTS: Of the 12,686 genes analyzed, 25 known genes were increased in expression (>4-fold) in GO orbital tissues, whereas 11 genes were decreased (>4-fold). Up-regulated genes, confirmed by quantitative RT-PCR, included secreted frizzled-related protein-1 (sFRP-1; 18.5-fold) and several adipocyte-related genes, including peroxisome proliferator activated receptor-gamma (44.1-fold) and adiponectin (25-fold). Treatment in vitro of GO orbital preadipocytes with recombinant sFRP-1 (100 nm) significantly increased adiponectin (2.0-fold; P < 0.05), leptin (7-fold; P < 0.002), and TSH receptor mRNA (13-fold; P < 0.003) levels and enhanced Oil red-O staining in the cultures. CONCLUSIONS: These results support the concept that orbital adipogenesis is enhanced in GO and suggest that elevated levels of sFRP-1 in the GO orbit may be involved in stimulating this pathogenic process.

Evidence for enhanced adipogenesis in the orbits of patients with Graves' ophthalmopathy.

The signs and symptoms of Graves' ophthalmopathy (GO) result from increased volume of the orbital contents, including adipose, connective, and extraocular muscle tissues. We wanted to determine whether the expanded adipose tissue volume might be in part attributable to de novo adipogenesis. We measured levels of mRNA encoding leptin, adiponectin, peroxisome proliferator-activated receptor gamma (PPAR gamma), preadipocyte factor-1, and TSH receptor (TSHr) genes in orbital adipose tissues from GO patients (n = 22) and normal individuals (n = 18) and in orbital preadipocyte cultures derived from GO patients (n = 6) and normal subjects (n = 3) using quantitative real-time RT PCR. We found increased leptin, adiponectin, PPAR gamma, and TSHr expression in GO compared with normal orbital tissue samples, with positive correlations in the GO tissues between TSHr and leptin, adiponectin and PPAR gamma. In vitro differentiation of GO and normal preadipocytes resulted in enhanced adiponectin, leptin, and TSHr expression, with greater expression of the latter two genes in the GO cultures. These results suggest that de novo adipogenesis within orbital tissues with parallel enhanced expression of TSHr may be important in the pathogenesis of GO, and that potential therapies for GO might include inhibition of the adipogenic pathway.