High affinity mAb infusion can enhance maximum affinity maturation during HIV Env immunization.

Antigen-specific antibody infusion is known to enhance or suppress germinal center (GC) responses depending on the affinity of the infusion. We hypothesized that infusing monoclonal antibodies (mAbs) of escalating affinity during an immunization regimen may progressively escalate selection pressure on competing B cells, increasing their affinity. To test this, we immunized mice with HIV envelope gp120 and infused CD4 binding-site (CD4bs)-specific mAbs. While mAb infusion reduced somatic hypermutation (SHM) and affinity in most CD4bs-specific B cells, a sub-population was identified with greater SHM and affinity than control. High-throughput sequencing of plasma cells revealed that CD4bs-specific plasma cells possessed elevated SHM after mAb infusion, with phylogenetic tree topology that suggested more rapid differentiation. We therefore conclude, in accordance with other studies, that high-affinity mAb infusion primarily suppresses recruitment of most competing B cells but can increase and expedite affinity maturation of certain epitope-specific B cells.

Sinus augmentation using rhBMP-2/ACS in a mini-pig model: Influence of an adjunctive ceramic bone biomaterial.

BACKGROUND: Recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge carrier (rhBMP-2/ACS) has been shown to support significant bone formation when used to augment the maxillary sinus for implant dentistry. Nevertheless, bone biomaterials have been suggested to extend rhBMP-2/ACS with limited support of the merits of such approaches. OBJECTIVES: To evaluate local bone formation/dental implant osseointegration following implantation of rhBMP-2/ACS combined with a ceramic bone biomaterial using a mini-pig sinus augmentation model. METHODS: Twelve adult Göttingen mini-pigs received rhBMP-2/ACS (rhBMP-2 adjusted to 0.43 mg/cc) alone or combined with an off-the-shelf biphasic ceramic (15%/85% HA/ß-TCP) biomaterial at 3:1, 1:1 and 1:3 ratios randomized to contra-lateral maxillary sinus sites yielding rhBMP-2/ACS fractions of 100%, 75%, 50% and 25%, respectively. A 4-cc implant volume was used for all sites. Two threaded dental implants (ø4.0 × 11.5 mm) were placed at each site. The animals were euthanized at 8 weeks for histologic analysis. RESULTS: Surgical execution and healing were generally uneventful, infraorbital local swelling was observed in all animals until suture removal. rhBMP-2/ACS combined with the ceramic biomaterial did not significantly enhance local bone formation (range 9.0 ± 1.5 to 9.7 ± 2.1 mm) compared with rhBMP-2/ACS alone (8.6 ± 1.1 mm; p > 0.05). Variations in rhBMP-2/ACS to ceramic matrix ratios yielding rhBMP-2 doses approximating 0.4, 0.9, 1.3 and 1.7 mg/sinus did not appreciably influence bone formation/osseointegration. CONCLUSIONS: Whereas rhBMP-2/ACS supports significant bone formation/osseointegration in the mini-pig sinus augmentation model and thus appears an effective alternative for sinus augmentation procedures, adding a ceramic biomaterial to rhBMP-2/ACS does not produce meaningful biological advantages.

Sinus augmentation using a mini-pig model: Effect of ceramic and allogeneic bone biomaterials.

BACKGROUND: Present clinical practice broadly relies on off-the-shelf allogeneic, xenogeneic or synthetic bone biomaterials in support of sinus augmentation. Also, recombinant human bone morphogenetic protein-2 in an absorbable collagen sponge carrier (rhBMP-2/ACS) has been shown to support clinically relevant bone formation when used to augment the maxillary sinus. OBJECTIVES: To evaluate local bone formation/dental implant osseointegration following implantation of two particulate bone biomaterials using the mini-pig sinus augmentation model. METHODS: Nine adult Göttingen mini-pigs were used for evaluation of a biphasic ceramic (15%/85% HA/ß-TCP) and an allogeneic mineralized bone biomaterial. Treatments randomized to contralateral sinus sites included sham-surgery (control) and biomaterials. Two threaded dental implants (ø4.0 × 11.5 mm) were placed at each sinus site. The animals were euthanized at 8 weeks for histologic analysis. RESULTS: Execution of the surgical protocol and healing was unremarkable. Limited infraorbital swelling was observed until suture removal. The biphasic ceramic and allogeneic bone biomaterials produced significantly increased bone formation (5.2 ± 1.9 mm and 4.9 ± 1.6 mm vs. 2.6 ± 0.5 mm, p < 0.05) and osseointegration (18.0 ± 6.0% and 25.1 ± 18.2% vs. 10.1 ± 8.0%, p < 0.05) over the sham-surgery control. No significant differences were observed between biomaterials. CONCLUSIONS: Implantation of biphasic ceramic or allogeneic bone biomaterials enhances bone formation in the mini-pig maxillary sinus, however, dental implant bone support is incomplete resulting in overall limited osseointegration.

Effect of rhBMP-2 dose on bone formation/maturation in a rat critical-size calvarial defect model.

BACKGROUND: Application of recombinant human bone morphogenetic protein-2 (rhBMP-2) has been associated with significant adverse events in craniofacial settings, including swelling and seroma formation. Recent work has demonstrated an inverse relationship between bone formation/maturation and rhBMP-2 dose, frequency/severity of adverse events increasing with rising dose. OBJECTIVE: The objective of this study was to determine the most effective dose for rhBMP-2 soak-loaded onto an absorbable collagen sponge (ACS) carrier for bone formation/maturation using an established defect model. METHODS: One hundred sixty-eight outbred male Sprague-Dawley rats, age 11-13 weeks, weight 325-375 g randomized into seven groups of 24 subdivided into groups of eight, were used to provide radiographic and light microscopy observations of bone formation/maturation and aberrant healing events at 2, 4 and 8 weeks following application of rhBMP-2/ACS into critical-size, ø8-mm, through-through, calvarial osteotomy defects for a dose of 1.25, 2.5, 5.0, 10.0 and 20.0 µg rhBMP-2/defect, or serve as ACS or sham-surgery controls. RESULTS: rhBMP-2 dosages ≥ 2.5 µg/defect showed histological defect closure >90% within 2 weeks, and complete resolution within 4 weeks. Adverse healing events including swelling, excessive bone formation or seroma formation could not be determined with certainty in this defect model. Notably ACS control sites showed complete defect closure at the 8-week healing interval. CONCLUSIONS: rhBMP-2/ACS accelerates local bone formation in the rat critical-size through-through calvarial defect model once reaching an osteoinductive dose threshold. This threshold may already be reached at a 1.25-/2.5-µg dose in this model. No further enhancement to bone formation/maturation may be observed adding rhBMP-2 above the 2.5-µg dose. The 1.25-20.0 µg dose range did not invoke appreciable aberrant healing events.

Evaluation of a compression resistant matrix for recombinant human bone morphogenetic protein-2.

BACKGROUND: Previous studies document the therapeutic potential of recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS) carrier for indications in the axial and appendicular skeleton. Nevertheless, the ACS does not comprise structural integrity to adequately support bone formation for onlay indications. The objective of this study was to evaluate local bone formation and osseointegration following surgical implantation of rhBMP-2 soak-loaded onto a compression resistant matrix (CRM). METHODS: Routine, contralateral, critical-size, supraalveolar, peri-implant defects in five adult male Hound Labrador mongrel dogs received 0.8 mg rhBMP-2 soak-loaded onto either the ACS (benchmark control) or a CRM (collagen/ß-TCP/hydroxyapatite) followed by submerged wound closure for primary intention healing. The animals were euthanized at 8 weeks for histologic/histometric evaluation. RESULTS: Healing was uneventful albeit considerable initial swelling was observed for either treatment. Sites receiving rhBMP-2/CRM showed significantly increased bone area (20.0 ± 0.9 versus 12.3 ± 2.6 mm(2) , p = 0.03) and bone density (24.1 ± 1.4% versus 14.6 ± 2.0%, p = 0.04) compared with those receiving rhBMP-2/ACS. There were no significant differences between treatments for new bone height and osseointegration. Woven and lamellar trabecular bone lined with abundant osteoid was observed for all sites. Inconsistent cortex formation confirmed the immature nature of the newly formed bone. Seroma formation was observed for both treatments (80-100% of the animals/implants). Sites receiving rhBMP-2/CRM showed residual ceramic granules undergoing biodegradation, including accumulation of foamy macrophages. CONCLUSIONS: rhBMP-2/CRM supports bone formation of clinically relevant geometry. Longer observation intervals as well as dose variations appear necessary to capture maturation of the newly formed bone, elimination of residual ceramic granules and resolution of seroma formation(s).

Sinus augmentation using rhBMP-2/ACS in a mini-pig model: relative efficacy of autogenous fresh particulate iliac bone grafts.

BACKGROUND: Implant dentistry in the posterior maxilla often requires bone augmentation. The gold standard, autogenous bone graft, requires additional surgery with associated morbidity, while bone biomaterials may not support relevant bone formation. Recombinant human bone morphogenetic protein-2 (rhBMP-2) in an absorbable collagen sponge (ACS), however, induces significant, clinically relevant bone formation in several settings including the maxillary sinus floor. OBJECTIVE: The objective of this study was to compare local bone formation and osseointegration following maxillary sinus augmentation using rhBMP-2/ACS or a particulate autogenous cancellous bone graft obtained from the iliac crest in conjunction with immediate placement of dental implants. MATERIALS AND METHODS: Bilateral sinus augmentation using an extraoral approach including rhBMP-2 (0.43 mg/ml)/ACS or the autogenous bone graft, alternated between left and right sinus cavities in five adult male Yucatan mini-pigs, was performed. Two 12-mm dental implants were inserted into the sinus wall protruding approximately 8 mm into the sinus cavity. Surgical sites were closed and sutured in layers; block biopsies collected for histometric analysis at 8 weeks. RESULTS: rhBMP-2/ACS induced bone of significantly greater and consistent quality compared with the iliac crest autogenous bone graft; bone density averaging 51.9 ± 3.0% vs. 32.9 ± 2.5% (P = 0.01). However, there were only numerical differences in augmented bone height (9.3 ± 0.5 vs. 8.6 ± 0.7 mm) and bone-implant contact (37.4 ± 3.0% vs. 30.7 ± 5.9%) between treatments. CONCLUSION: rhBMP-2/ACS induces bone of superior quality compared with an iliac crest particulate autogenous cancellous bone graft when used for maxillary sinus augmentation, and should perhaps be considered the new standard for this indication.

Recombinant human bone morphogenetic protein 2 combined with an osteoconductive bulking agent for mandibular continuity defects in nonhuman primates.

PURPOSE: Recombinant human bone morphogenetic protein 2 (rhBMP-2) is an option for reconstructing mandibular continuity defects. A challenge of this technique is the need to maintain sufficient space to avoid compression of the defect. A compression-resistant matrix (CRM) provides a bulking agent that provides support during the bone formation phase. MATERIALS AND METHODS: Thirteen Rhesus Macaque monkeys were used to evaluate different forms of an osteoconductive bulking agent compared with an absorbable collagen alone placed into a critical-sized mandibular defect. A total of 5 groups (26 defects) were evaluated: group A, rhBMP-2/absorbable collagen sponge (ACS) (1.5 mg/mL); group B, rhBMP-2/ACS with ceramic granules (15% hydroxyapatite/85% ß-tricalcium phosphate) at 1.5 mg/mL; group C, rhBMP-2 (2.0 mg/mL) with a CRM; group D, rhBMP-2 (0.75 mg/mL) with a CRM; and group E, a CRM alone. RESULTS: Histology and micro computed tomography were used to evaluate and compare new bone formation in the defects. The reconstructed bone was evaluated with regard to the new bone formation, residual voids, and density. Animals treated with the CRM and rhBMP-2 at 2.0 mg/mL (group C) showed significantly higher amounts of new bone formation, bone density, and reduced voids when compared with rhBMP-2 and ACS (1.5 mg/mL) (P < .05). CONCLUSION: The carrier system CRM combined with rhBMP-2 and a reconstruction plate results in significantly higher bone density and better space maintenance than rhBMP-2 combined with ACS in a nonhuman primate mandibular bone repair model.

Temporal exposure to chondrogenic factors modulates human mesenchymal stem cell chondrogenesis in hydrogels.

Tissue engineering utilizes scaffolds containing chondrogenic cells to promote cartilage development at a clinically relevant scale, yet there remains a limited understanding of the optimal conditions for inducing differentiation and matrix production. We investigated how cell density and temporal exposure to chondrogenic factors impacted chondrogenesis of human mesenchymal stem cells (hMSCs) encapsulated in poly(ethylene glycol) diacrylate hydrogels. We found maximal proteoglycan and collagen production in constructs seeded between 10 and 25 × 10(6) cells/mL. Matrix deposition was significantly less per cell in constructs seeded at either higher or lower densities, indicating that paracrine communications may remain important despite loss of direct cell-cell contact. In vitro chondrogenesis of hMSCs was first accomplished using pellet cultures and a defined medium containing transforming growth factor (TGF)-ß1 and dexamethasone. The differentiation of hMSCs in hydrogels also required initial exposure to TGF-ß1, with no chondrogenic matrix produced in its absence. If TGF-ß1 was initially included for at least 7 days, its removal impacted collagen production per cell but also lead to an increase in cell number, such that total collagen deposition was equivalent to controls when TGF-ß1 was included for at least 3 weeks. Further, proteoglycan content per construct was higher at 6 weeks after removal of TGF-ß1 at any time. In contrast to TGF-ß1, dexamethasone was not required for chondrogenesis of hMSCs in hydrogels: there was no difference in matrix deposition between hydrogels cultured with or without dexamethasone. Further, without dexamethasone, SOX9 gene expression was higher during early chondrogenesis and there was a significant reduction in collagen I deposition, suggesting that a more hyaline cartilage phenotype is achieved without dexamethasone. Collagen content at 6 weeks was lower if dexamethasone was excluded after the first 7 days, but was equivalent to control if dexamethasone was included for 2 weeks or longer. Proteoglycan deposition was unaffected by dexamethasone exclusion. These results indicate that modulating exposure to TGF-ß1 is beneficial for cell survival/proliferation and matrix production from hMSCs in hydrogels, and that not only is dexamethasone dispensable but also its exclusion may be advantageous for forming hyaline cartilage.

Design and characterization of poly(ethylene glycol) photopolymerizable semi-interpenetrating networks for chondrogenesis of human mesenchymal stem cells.

Mesenchymal stem cells (MSCs) are used extensively in cartilage tissue engineering. We have developed a photopolymerizable poly(ethylene glycol diacrylate) (PEGDA) and poly(ethylene glycol) (PEG) semi-interpenetrating network that facilitates the in vitro chondrogenesis of human MSCs (hMSCs). Network parameters were altered and tested for their effects on subsequent matrix elaboration. The mesh size, calculated for each network based on equilibrium swelling ratios, was larger with lower PEGDA:PEG ratios and with higher PEGDA molecular weight. Changes in xi correlated with changes in extracellular matrix content and deposition in hMSC-seeded networks cultured in vitro for 6 weeks in defined chondrogenic medium. Networks constructed with PEGDA (6 kDa) and PEG (88 kDa) at 1:2 displayed intercellular deposition of proteoglycan. Furthermore, their proteoglycan contents were significantly higher than with PEGDA (6 kDa) hydrogels constructed without the PEG component and those constructed at a PEGDA:PEG ratio of 2:1, which both exhibited pericellular proteoglycan deposition. However, networks constructed with PEGDA (12 and 20 kDa) and PEG (88 kDa) exhibited intercellular deposition of proteoglycan regardless of the ratio employed. Collagen content was lower in networks constructed with PEGDA (12 and 20 kDa) and PEG (88 kDa) at a ratio of 1:2 than in those fabricated at the same PEGDA molecular weights at a ratio of 2:1. This study demonstrated that semi-interpenetrating network parameters influence not only extracellular matrix content, but also the deposition of the matrix molecules by hMSCs undergoing chondrogenesis. It is important that these parameters be considered carefully when creating scaffolds for tissue-engineered cartilage.