Engineering of immunoinstructive extracellular matrices for enhanced osteoinductivity

The increasing recognition of the contribution of the immune system to activate and prime regeneration implies that tissue engineering strategies and biomaterials design should target regulation of early immunological processes.We previously proposed the cell-based engineering and devitalization of extracellular matrices(ECMs)as a strategy to generate implant materials delivering custom-defined signals.Here,in the context of bone regeneration,we aimed at enhancing the osteoinductivity of such ECMs by enriching their immunomodulatory factors repertoire.Priming with IL1βa cell line overexpressing BMP-2 enabled engineering of ECMs preserving osteoinductive signals and containing larger amounts of angiogenic(VEGF)and pro-inflammatory molecules(IL6,IL8 and MCP1).Upon implantation,these IL1β-induced materials enhanced processes typical of the inflammatory phase(e.g.,vascular invasion,osteoclast recruitment and differentiation),leading to‘regenerative’events(e.g.,M2 macrophage polarization)and ultimately resulting in faster and more efficient bone formation.These results bear relevance towards the manufacturing of potent off-the-shelf osteoinductive materials and outline the broader paradigm of engineering immunoinstructive implants to enhance tissue regeneration.

Sudoku of porous, injectable calcium phosphate cements – Path to osteoinductivity

With the increase of global population,people’s life expectancy is growing as well.Humans tend to live more active lifestyles and,therefore,trauma generated large defects become more common.Instances of tumour resection or pathological conditions and complex orthopaedic issues occur more frequently increasing necessity for bone substitutes.Composition of calcium phosphate cements(CPCs)is comparable to the chemical structure of bone minerals.Their ability to self-set and resorb in vivo secures a variety of potential applications in bone regeneration.Despite the years-long research and several products already reaching the market,finding the right properties for calcium phosphate cement to be osteoinductive and both injectable and suitable for clinical use is still a sudoku.This article is focused on injectable,porous CPCs,reviewing the latest developments on the path toward finding osteoinductive material,which is suitable for injection.

Simple application of adipose-derived stem cell-derived extracellular vesicles coating enhances cytocompatibility and osteoinductivity of titanium implant

Surface modification using bioactive molecules is frequently performed to improve the biological properties of medical metal biomaterial titanium(Ti)implants.Developmental evidence suggests that mesenchymal stem cell-derived extracellular vesicles(MSC-EVs)served as potent bioactive component.As a subset of MSC EV,adipose-derived stem cel-derived extracellular vesicles(ADSC-EVs)could be obtained from abundant adipose tissue.Meanwhile,it possesses multiple re-generative properties and might be used to endow biological activities to medical Ti implant.Here,we present a simple ADSC-EV coating strategy based on physisorption of fibronectin.This ADSC-EV functionalized Ti implants(EV-Ti)revealed enhanced osteoblast compatibility and osteoinduc-tive activity.Cell spreading area of EV-Ti group was 1.62-and 1.48-fold larger than that of Ti group after 6 and 12 h of cell seeding,respectively.Moreover,EV-Ti promoted alkaline phosphatase,col-lagen 1 and osteocalcin gene expression in osteoblast by 1.51-,1.68-and 1.82-fold compared with pristine Ti,respectively.Thus,the MSC-EVs modification method reported here provide a clinically translatable strategy to promote the bioactivity of Ti implants.

Ectopic Bone Formation in vivo Induced by a Novel Synthetic Peptide Derived from BMP-2 Using Porous Collagen Scaffolds

To investigate the osteoinductive and ectopicly osteogenic effects of a novel peptide P24 derived from bone morphogenetic protein 2 （BMP2）, biodegradable collagen scaffolds （CS） were used to load BMP-2-derived peptide solutions with different concentrations （0.4 mg peptide/CS, 0.1 mg peptide/CS and pure CS, respectively）, and the implants were implanted into muscular pockets on the back of Wistar rats. Radiographs and histological analysis were performed to evaluate the ectopic bone effects. Active ectopic bone formation was seen in both groups containing the peptide at different concentration （0.4 mg and 0.1 mg）, whereas no bone formation and only fibrous tissue was seen in the pure CS group. The new bone formation induced by the peptide P24 displayed a dose-dependent and time-dependent efficiency. The new bone formation in the 0.4 mg peptide/CS group significantly increased than that of the 0.1 mg peptide/CS group. This novel BMP-2-derived peptide had excellent osteoinductive and ectopicly osteogenic properties which were similar to those of BMP2.

Porous Calcium Phosphate Ceramic Scaffolds for Tissue Engineering

This study examined the biological response of two porous calcium phosphate ceramics, hydroxyapntite （ HA ） and hydroxyapaptite/β-tricalcium phosphate （ HA/β- TCP ） scaffolds. Three different cell types , a human osteoblastic cell line （ HOS ） , primary human osteoblasts （HOB） and human mesenehymal stem cells （MSCs）, were used to examine biocompatibility and osteoinductive capacity. The experimental results showed both materials were highly biocompatible and proliferation was significantly greater on pure HA （ P 〈 0.01 ）, with a peak in proliferation at day 7. Protein levels were significantly higher （ P 〈 0.05） than the control Thermanox（ TMX （ tm） ） for both test materials. Osteoinduction of MSCs was observed on both test materials, with cells seeded on HA/ β-TCP showing greater alkaline phosphatase activity compared to HA alone, indicating an enhancement in osteoinductive property. Both materials show good potential for use as tissue engineered scaffolds.

Osteogenesis Capacity of a Novel BMP/α-TCP Bioactive Composite Bone Cement

To improve the osteogenesis ability of a-tricalcium phosphate (α-TCP) bone cement, a novel BMP/ α-TCP composite bone cement was prepared. By measuring the setting time and compressive strength, the hydration characteristic of bone cement was evaluated. Animal experiments including histological observation, radiographic investigation as well as digital image analyses reveal the difference of osteogenesis ability among BMP,a-TCP bone cement and BMP/α-TCP composite bone cement. Results show that α-TCP bone cement possesses excellent hydration and setting properties as well as high mechanical property. Comparison experiments show that BMP/ α-TCP composite bone cement has a stronger osteogenesis ability. The gross observation of the implant site does not exhibit any inflammation or necrosis. Histological analyses reveal that the material has good osteointegration with host bone, and new bone formation is detected within the materials, which are degrading. Strong osteogenesis ability of the composite is due to not only the excellent osteoconductive potential but also the osteoinductive potential contributed by active BMP releasing and the material degradation. Large skull defect could be well-healed by filling BMP/α-TCP composite bone cement. This novel material proves itself to be an absorbable and bioactive bone cement with an osteogenesis ability. Key words α-tricalcium phosphate (α-TCP) - bone morphogenetic proteins (BMP) - bone cement - osteogenesis - osteoinductivity - bone tissue engineering Funded by 863 Hi-Tech Research and Development Program of China (2002AA326080) and the Fund for Outstanding Young Teacher of the Education Ministry of China(2002123)

Immobilization of RGD Peptide onto the Surface of Apatite-Wollastonite Ceramic for Enhanced Osteoblast Adhesion and Bone Regeneration

The arginine-glycine-aspartic （RGD） acid peptide was grafted to the surface of apatitewollastonite （AW） ceramic in an effort to improve its cell adhesion, proliferation and osteoinduction. RGD peptide was covalently immobilized onto the surface of AW ceramic via the synthetic cross linker AA.PTS-E and 1-ethyl-3-（3-dimethylaminopropyl）carbodiimide （EDC）. The modified surfaces were characterized by attenuated total reflectance Fourier transform infrared spectroscopy （ATR-FTIR） and X-ray photoelectron spectroscopy （XPS）. The chemical analysis indicated that RGD peptide had been immobilized onto the AW surface successfully. The growth of osteoblast-like cells （MG63） showed that modifying the AW surface with RGD peptide enhanced the cell adhesion and proliferation. And the histological evaluation of RGD-AW showed that the bone regeneration and remodeling process were significantly enhanced compared to the original AW ceramics after 2, 4 and 8 weeks implantation in rabbit＇s femoral condyles.

In Vitro Characterizations of PLLA/β-TCP Porous Matrix Materials and RMSC-PLLA-β-TCP Composite Scaffolds

To develop a novel degradable poly (L-lactic acid)/β-tricalcium phosphate (PLLA/β-TCP) bioactive materials for bone tissueengineering, β-TCP powder was produced by a new wet process. Porous scaffolds were prepared by three steps, i.e. solventcasting, compression molding and leaching stage. Factors influencing the compressive strength and the degradation behaviorof the porous scaffold, e.g. weight fraction of pore forming agent-sodium chloride (NaCl), weight ratio of PLLA: β-TCP,the particle size of β-TCP and the porosity, were discussed in details. Rat marrow stromal cells (RMSC) were incorporatedinto the composite by tissue engineering approach. Biological and osteogenesis potential of the composite scaffold weredetermined with MTT assay, alkaline phosphatase (ALP) activity and bone osteocalcin (OCN) content evaluation. Resultsshow that PLLA/β-TCP bioactive porous scaffold has good mechanical and pore structure with adjustable compressive strengthneeded for surgery. RMSCs seeding on porous PLLA/β-TCP composite behaves good seeding efficacy, biocompatibility andosteoinductive potential. Osteoprogenitor cells could well penetrate into the material matrix and begin cell proliferation andosteogenic differentiation. Osseous matrix could be formed on the surface of the composite after culturing in vitro. It isexpected that the PLLA/β-TCP porous composites are promising scaffolds for bone tissue engineering in prosthesis surgery.

Heterotopic ossification after the use of recombinant human bone morphogenetic protein-7

AIM To present the incidence of heterotopic ossification after the use of recombinant human bone morphogenetic protein-7(rhB MP-7) for the treatment of nonunions.METHODS Bone morphogenetic proteins(BMPs) promote bone formation by auto-induction. Recombinant human BMP-7 in combination with bone grafts was used in 84 patients for the treatment of long bone nonunions. All patients were evaluated radiographicaly for the development of heterotopic ossification during the standard assessment for the nonunion healing. In all patients(80.9%) with radiographic signs of heterotopic ossification, a CT scan was performed. Nonunion site palpation and ROM evaluation of the adjacent jointswere also carried out. Factors related to the patient(age, gender), the nonunion(location, size, chronicity, number of previous procedures, infection, surrounding tissues condition) and the surgical procedure(graft and fixation type, amount of rhB MP-7) were correlated with the development of heterotopic ossification and statistical analysis with Pearsons χ~2 test was performed.RESULTS Eighty point nine percent of the nonunions treated with rh BMP-7, healed with no need for further procedures. Heterotopic bone formation occurred in 15 of 84 patients(17.8%) and it was apparent in the routine radiologi-cal evaluation of the nonunion site, in a mean time of 5.5 mo after the rh BMP-7 application(range 3-12). The heterotopic ossification was located at the femur in 8 cases, at the tibia in 6, and at the humerus in οne patient. In 4 patients a palpable mass was present and only in one patient, with a para-articular knee nonunion treated with rhB MP-7, the size of heterotopic ossification affected the knee range of motion. All the patients with heterotopic ossification were male. Statistical analysis proved that patient's gender was the only important factor for the development of heterotopic ossification(P = 0.007). CONCLUSION Heterotopic ossification after the use of rh BMP-7 in nonunions was common but it did not compromise the final clinical outcome in most cases, and affected only male patients.

Tissue Response of an Osteoinductive Bioceramic in Bone Defect Rabbit Model

HA/TCP and HA rods （φ5 mm×10 mm） were made for implantation in New Zealand white rabbit with different condition. Sixty three rabbit were divided into three groups： group 1 （n=18）, group 2 （n=27） and group 3 （n=18）. In group 1, 10 mm radius was defected, and one HA/TCP rod was implanted in the muscle a distant away from the bone defect area. In group 2, also, 10 mm radius was defected, one HA rod was implanted in the muscle a distant away from the bone defect area. In group 3, two HA/TCP rods were implanted in the dorsal muscle of the rabbit with bone intact. Histological observation showed that in group 1, some new bone was found only two months after implantation （n=2）, and obvious immature woven bone could be observed in these bioceramics from the 3rd month on. However, in group 3, bone began to be found 6 months after implantation （n=2）. In group 2, we could not find any bone tissue up to 9 month’s observation. These results suggest that, first, the bone defect model could significantly accelerate bone formation at non-osseous sites in rabbits; second,. HA/TCP bioceramics were confirmed with osteoinductive property while HA bioceramics without osteoinductive property nearly. Thus, bone defect might be a good animal model for further researches for osteoinductive bioceramics.

The Clinical Application of Human Bone Matrix Gelatin

This paper reports the results of 24 cases of bone defect resulting from bone tumor or tumor condition excision, and of posterior spinal fusion, treated by human bone matrix gelatin. The success rate of bone defect repair and spinal fusion is 91. 67 %. The results suggest that human bone matrix gelatin has. excellent osteoinductive effect and is ideal substitute for bone autografts.

The Role of Microstructure of Highly Purified Beta-Tricalcium Phosphate for Osteoinduction in Canine Dorsal Muscles

Porous β-tricalcium phosphate (TCP) displays osteoinductivity in certain animals in the absence of osteoinductive agents. We evaluated whether the microstructure may be an important determinant of osteoinduction, and also investigated how bone formation was promoted using β-TCP combined with bone marrow aspirates. We prepared two types of β-TCP, namely, β-TCP A, which possessed interconnected macropores and micropores, and β-TCP B, which possessed macropores but had less detectable micropores. These were implanted with or without marrow in canine muscles. Bone formation and the resorption of each β-TCP implant were evaluated histologically. Newly formed bone began to appear at day 42 in the implants of β-TCP A alone, but the implants of β-TCP B alone did not show any bone formation by day 42. Meanwhile, bone formation was already evident on day 14 by loading with bone marrow aspirates with or without micropores. By immunohistochemistry, the number of cathepsin K-positive cells (osteoclasts) increased as time passed in the implants of β-TCP A alone, while the number of the osteoclasts did not change obviously in the implants of β-TCP B alone from day 14 to 56. Reticular fibrils were evident within the β-TCP A, and were barely observed in the β-TCP B in the silver impregnation. The present result would bring about the possible role to enhance the importance of the surface microstructure for the better osteoinductivity. Our findings suggest that the combination of porous β-TCP and bone marrow facilitates bone formation.

Osteoinduction and Osteoconduction with Porous Beta-Tricalcium Phosphate Implanted after Fibular Resection in Humans

Osteoinductive properties of β-TCP remain unknown in humans. It is important to improve the bone grafts which have been the standard treatment for bone defect due to their biocompatibility and bone-healing properties. The purpose of this study was to radiologically clarify the bone forming property of β-TCP by evaluating the replacement of β-TCP by newly formed bone in the defect after fibular resection and to examine the histological features of a β-TCP specimen three months after grafting. Radiographs of 17 patients who underwent β-TCP grafting were evaluated. Osteoinductive and osteoconductive properties were assessed by examining bone formation from the remnant fibula, periosteum, and β-TCP alone. In one case, β-TCP was removed later because of postoperative complications and was evaluated histologically. Twenty two of 34 sites between the remnant fibula and β-TCP had achieved good bone regeneration. Five of 14 sites between the periosteum and β-TCP had achieved good bone regeneration. We found immature but evident bone formation in three cases with no osseous and periosteal sites. Histological analysis revealed bone formation on the outer macropore surface of β-TCP. Some blood vessels formed in the macropores expressed CD31 and CD34, while a few lymphatic vessels expressed CD34 and podoplanin. Thus, the osteoinductive ability of β-TCP alone was demonstrated in humans radiographically for the first time. The histological morphology of β-TCP was demonstrated at an early stage after grafting in humans.

Induced Bone by Calcium Ohosphate (Ca-P) Ceramics after 2-years Implantation

The changes of induced bone with four kinds of Ca-P ceramics after 2 years implantation in the dorsal muscles of rabbits were investigated. After 2 years implantation, mature bone （with a lamellar structure after the remodeling process） with bone marrow was observed in HA1100 （HA, micro＋macro porous）, HA900 （HA, micro＋macro porous） and BCP （HA/TCP=7：3, micro＋macro porous）, and no bone formation was observed in HA1200 （HA, macro porous） ceramics. The induced bone neither disappeared nor grew uncontrollably, and was seen inside the pores of the implants or on the outer surface under the fibrous layer after 2 years. No bone formation was observed in the soft tissues distant from the implants. These results indicate that heterotopic bone formation induced by Ca-P materials did not give rise to uncontrolled growth over time, and the induced bone is limited to the inside or around the implants. Moreover, ectopic bone formation on the outer surface of the implants was detected in rabbits after 2 years.

Dual-Wavelength Photosensitive Nano-in-Micro Scaffold Regulates Innate and Adaptive Immune Responses for Osteogenesis

The immune response of a biomaterial determines its osteoinductive effect.Although the mechanisms by which some immune cells promote regeneration have been revealed,the biomaterial-induced immune response is a dynamic process involving multiple cells.Currently,it is challenging to accurately regulate the innate and adaptive immune responses to promote osteoinduction in biomaterials.Herein,we investigated the roles of macrophages and dendritic cells(DCs)during the osteoinduction of biphasic calcium phosphate(BCP)scaffolds.We found that osteoinductive BCP directed M2 macrophage polarization and inhibited DC maturation,resulting in low T cell response and efficient osteogenesis.Accordingly,a dual-targeting nano-in-micro scaffold(BCP loaded with gold nanocage,BCP-GNC)was designed to regulate the immune responses of macrophages and DCs.Through a dual-wavelength photosensitive switch,BCP-GNC releases interleukin-4 in the early stage of osteoinduction to target M2 macrophages and then releases dexamethasone in the later stage to target immature DCs,creating a desirable inflammatory environment for osteogenesis.This study demonstrates that biomaterials developed to have specific regulatory capacities for immune cells can be used to control the early inflammatory responses of implanted materials and induce osteogenesis.

Experimental Study of Porous Beta-tricalcium Phosphate and Bone Morphogenetic Protein/porous Beta-tricalcium Phosphate Complex in Rabbit Mandibular Reconstruction

We investigated the osteogenic characteristic and biodegradation behavior of porous beta-tricalcitum phosphate （ β- TCP ） and bone morphogenetic protein/beta-tricalcium phosphate （ BMP/ β- TCP ） complex in mandibular reconstruction and evaluated the advantages of BMP / β- TCP complex in repairing bone defects. Bone defects created in the lower margin of bilateral mandible bodies in 12 rabbits were repaired with β- TCP （ control group ） and BMP/ β- TCP complex （ experimental group ） respectively. The rabbits were euthanized after 2, 4, 8 and 12 weeks and examined by macroscopy, radiography, histology, histomorphometry and image analysis. 2 weeks after surgery, ossftcation of newly-generated tissue in BMP/ β- TCP complex group appeared and after 12 weeks, massive new bone and ossification maturation were seen. However, in β- TCP group without BMP , ossification was not found until 12 weeks after operation. Image analysis showed that bone regeneration rate of BMP/ β- TCP was 30%-40% higher and the degradation rate was 20%- 30% higher than that of β- TCP. Therefore, as a reconstructive material for bone defects, BMP/ β-TCP complex is superior to β-TCP and can be used in oral and maxillofacial surgery.

Local Application of Alendronate on <i>β</i>-Tricalcium Phosphate Accelerated Induction of Osteogenesis with Formation of Giant Osteoclast-Like Cell

Intrinsic osteoinductivity—the ability to induce bone formation in ectopic sites without addition of osteogenic factors has been reported in various porous materials. Tartrate-resistant acid phosphatase (TRAP)-positive osteoclast-like cells are thought to play an important role in material-induced osteoinduction. To investigate the influence of osteoclastic activity on intrinsic osteoinduction, we loaded alendronate (10–2 , 10–4 , and 10–6 M) onto porous β-tricalcium phosphate (β-TCP) blocks to inhibit osteoclastic activity, and evaluated osteoinductivity by implantation of the blocks into the dorsal muscles of adult beagle dogs. Alendronate-loaded porous β-TCP blocks increased both speed and amount of osteoinduction, as measured 4 weeks after implantation, with the 10–4 M alendronate-loaded β-TCP being especially active. This finding indicates that β-TCP loaded with 10–4 M alendronate might prove crucial in providing the desirable balance between the degradation rate of bone scaffolds and their osteoinductive replacement. Thus, material-induced osteoinduction may be controlled by local application of alendronate, establishing alendronate loading as a promising therapeutic approach.

One-step co-doping of ZnO and Zn^(2+)in osteoinductive calcium phosphate ceramics with synergistic antibacterial activity for regenerative repair of infected bone defect

How to endow bone grafts with long-term antibacterial activity and good bone regenerative ability to achieve the regenerative repair of infected bone defects has been the focus of the clinical treatment of osteomyelitis.The present study introduced a novel one-step route to realizing the co-doping of zinc oxide(ZnO)and zinc ion(Zn^(2+))in biphasic calcium phosphate(BCP)ceramics to utilize their synergistic antibacterial.Compared with the conventional BCP ceramics(BCP-Ca),the ZnO/Zn^(2+)co-doping ones(BCP-Zn)possessed strong antibacterial ability on E.coli and S.aureus as well as stimulated the osteogenic differentiation of bone marrow mesenchymal stem cells(BMSCs)effectively.The synergistic antibacterial mechanism of ZnO and Zn^(2+)was also investigated.BCP-Zn showed excellent osteoinductivity and angiogenesis at three months postoperatively in the canine intramuscular implantation model.Moreover,BCP-Zn exhibited excellent anti-infective ability and bone regenerative repair compared to BCP-Ca and control groups in the infected bone defect model of rat femur.Collectively,these findings suggest that the simultaneous introduction of ZnO/Zn^(2+)could have immense potential to expand the application of osteoinductive BCP ceramics in the regenerative repair of infected bone defects.

Continuously released Zn^(2+)in 3D-printed PLGA/β-TCP/Zn scaffolds for bone defect repair by improving osteoinductive and anti-inflammatory properties

Long-term nonunion of bone defects has always been a major problem in orthopedic treatment.Artificial bone graft materials such as Poly(lactic-co-glycolic acid)/β-tricalcium phosphate(PLGA/β-TCP)scaffolds are expected to solve this problem due to their suitable degradation rate and good osteoconductivity.However,insufficient mechanical properties,lack of osteoinductivity and infections after implanted limit its large-scale clinical application.Hence,we proposed a novel bone repair bioscaffold by adding zinc submicron particles to PLGA/β-TCP using low temperature rapid prototyping 3D printing technology.We first screened the scaffolds with 1 wt%Zn that had good biocompatibility and could stably release a safe dose of zinc ions within 16 weeks to ensure long-term non-toxicity.As designed,the scaffold had a multi-level porous structure of biomimetic cancellous bone,and the Young’s modulus(63.41±1.89 MPa)and compressive strength(2.887±0.025 MPa)of the scaffold were close to those of cancellous bone.In addition,after a series of in vitro and in vivo experiments,the scaffolds proved to have no adverse effects on the viability of BMSCs and promoted their adhesion and osteogenic differentiation,as well as exhibiting higher osteogenic and anti-inflammatory properties than PLGA/β-TCP scaffold without zinc particles.We also found that this osteogenic and anti-inflammatory effect might be related to Wnt/β-catenin,P38 MAPK and NFkB pathways.This study lay a foundation for the follow-up study of bone regeneration mechanism of Zn-containing biomaterials.We envision that this scaffold may become a new strategy for clinical treatment of bone defects.

Triple-functional bone adhesive with enhanced internal fixation,bacteriostasis and osteoinductive properties for open fracture repair

At present,effective fixation and anti-infection implant materials represent the mainstay for the treatment of open fractures.However,external fixation can cause nail tract infections and is ineffective for fixing small fracture fragments.Moreover,closed reduction and internal fixation during the early stage of injury can lead to potential bone infection,conducive to bone nonunion and delayed healing.Herein,we designed a bone adhesive with anti-infection,osteogenic and bone adhesion fixation properties to promote reduction and fixation of open fractures and subsequent soft tissue repair.It was prepared by the reaction of gelatin(Gel)and oxidized starch(OS)with vancomycin(VAN)-loaded mesoporous bioactive glass nanoparticles(MBGNs)covalently cross-linked with Schiff bases.Characterization and adhesion experiments were conducted to validate the successful preparation of the Gel-OS/VAN@MBGNs(GOVM-gel)adhesive.Meanwhile,in vitro cell experiments demonstrated its good antibacterial effects with the ability to stimulate bone marrow mesenchymal stem cell(BMSCs)proliferation,upregulate the expression of alkaline phosphatase(ALP)and osteogenic proteins(RunX2 and OPN)and enhance the deposition of calcium nodules.Additionally,we established a rat skull fracture model and a subcutaneous infection model.The histological analysis showed that bone adhesive enhanced osteogenesis,and in vivo experiments demonstrated that the number of inflammatory cells and bacteria was significantly reduced.Overall,the adhesive could promote early reduction of fractures and antibacterial and osteogenic effects,providing the foothold for treatment of this patient population.