Ignite Creation Date:
2024-10-25 @ 7:59 PM
Last Modification Date:
2024-10-26 @ 3:36 PM
Study NCT ID:
NCT06533150
Status:
NOT_YET_RECRUITING
Last Update Posted:
None
First Post:
2024-07-30
Brief Title:
Functionalized Bioink Delivering Biomolecules for the Treatment of Craniofacial Diseases
Sponsor:
None
Organization:
None
Study Overview
Official Title:
A Dopable Bioink for Augmented Tissue Engineering in Craniofacial Reconstruction Innovative Pipeline for Drug Design and Selective Delivery Through Functionalized Polymeric Nanoparticles
Status:
NOT_YET_RECRUITING
Status Verified Date:
2024-07
Last Known Status:
None
Delayed Posting:
No
If Stopped, Why?:
Not Stopped
Has Expanded Access:
No
If Expanded Access, NCT#:
N/A
Has Expanded Access, NCT# Status:
N/A
Acronym:
DART-CRAFT
Brief Summary:
The study aims to address the challenges of craniofacial bone reconstruction in pediatric and adult patients affected by congenital craniofacial malformations ie craniosynostosis trauma or tumors by developing an innovative biohybrid material with tunable rheological properties serving as a sealing agent and defect filler Craniectomycraniotomy procedures often leave bone defects that require cranioplasty to protect the underlying dura mater and the brain from physical insults Reconstruction of the viscerocranial skeleton poses additional challanges due to the complex anatomy of the facial skull and significant esthetic and functional demands on its reconstruction
The study plans to develop a mouldable biosynthetic gelatin-methacrylamide GelMA-based hydrogel complexed with functionalized Poly lactic-co-glycolic acid PLGA nanoparticles for drug delivery Osteoprogenitors cells including mesenchymal stromal cellsosteoblasts and monocytesosteoclasts will be isolated from bone tissue fragments of enrolled patients and peripheral blood sample respectively to obtain 2D and 3D cultures mimicking the in vivo bone environment High-throughput profiling of patients samples will identify druggable targets for the bioactive compounds to be released by the bioink In vitro validation will involve osteoprogenitor co-cultures derived from patients to assess uptake release dynamics biocompatibility immunogenicity and therapeutic effects of the developed complex The final goal will be to develop a pre-prototype tissue engineering biocomposite for craniofacial bone reconstruction
The study plans to develop a mouldable biosynthetic gelatin-methacrylamide GelMA-based hydrogel complexed with functionalized Poly lactic-co-glycolic acid PLGA nanoparticles for drug delivery Osteoprogenitors cells including mesenchymal stromal cellsosteoblasts and monocytesosteoclasts will be isolated from bone tissue fragments of enrolled patients and peripheral blood sample respectively to obtain 2D and 3D cultures mimicking the in vivo bone environment High-throughput profiling of patients samples will identify druggable targets for the bioactive compounds to be released by the bioink In vitro validation will involve osteoprogenitor co-cultures derived from patients to assess uptake release dynamics biocompatibility immunogenicity and therapeutic effects of the developed complex The final goal will be to develop a pre-prototype tissue engineering biocomposite for craniofacial bone reconstruction
Detailed Description:
To develop a mouldable biosynthetic collagen-based polymer matrix commercial gelatin-methacrylamide GelMA-based hydrogels will be biochemically modified to finely tune their pseudo-plasticity and yield stress and functionalized to implement drug delivery for improved biological properties The GelMA will be chemically enriched with adhesive molecules eg alginate-based or ion releasing inorganic compounds if needed to increase the adhesion to implantable materials used in craniofacial reconstruction thiol-based non- cleavage-type photoinitiators eg eosinY combined with triethanolamine and vinyl caprolactam to enable visible light-activated crosslinking and minimize the safety concerns of UV light Photo-crosslinking will be achieved through a portable visible light device 420-480 nm to induce the jellification of the bioink The physical properties morphology viscoelastic properties stiffness resistance to an applied stress of the different GelMA compounds will be analyzed according to standardized procedures The adhesive properties of the GelMA will be measured by lap-shear strength tests in dry conditions Once identified GelMA compounds showing the highest biological features this will be endowed with functionalized Poly lactic-co-glycolic acid PLGA-based nanoparticles NPs
To this aim biocompatible PLGA-Polyethylene glycol PEG NPs will be synthesized and functionalized with bis-sulfone for binding targeting moieties on the surface The synthesis involves PLGA activation PLGA-PEG conjugation bis-sulfone activation and PLGA-PEG-bis-sulfone conjugation The PLGAPEG ratio will be adjusted for the hydrophobicityhydrophilicity payload Bis-sulfone is used for selective and efficient PEGylation of proteins disulfide bond or to conjugate His-tagged proteinpeptides according to standardized methods to implement either specific cell targeting or antimicrobial agents High performance liquid chromatography HPLC will be used to validate the biosynthetic reaction Dynamic Light Scattering DLS Nanoparticle Tracking Analysis NTA and Scanning electron microscopy SEM will define the morphological and ultrastructural properties of the final construct Surface Plasmon Resonance will validate target recognition Then GelMA-NP hybrid compound assembly will be achieved through extrusion 3D printing according to standardized production pipelines The hydrogel and the NP suspension will be dispensed through separate nozzles in 2D and 3D patterns with 100-1000 nm feature sizes under pressure and temperature control Scalar NP concentrations will be used and then evaluated
NP integrity and release dynamics of these from GelMA will be studied by submerging the GelMA-NPs loaded with fluorescent compound with a bio-mimetic cell growth medium and quantifying the NPs released in the supernatant in time course experiments NP quantification will be performed using UV-Vis Spectroscopy Fluorescence Spectroscopy or particle counting such as NTA system NPs integrity will be studied with transmission electron microscopy TEM and DLS
Once biochemically characterized GelMA-NPs these will be used to deliver bioactive compounds identified through proteomics data as subsequently described
To validate the developed bio-ink in in vitro craniofacial disease models patients with craniofacial malformations undergoing cranial surgery will be selected and enrolled in the study Specifically paediatric patients affected by craniosynostosis and other inborn defects trauma or brain tumors undergoing cranial surgery will be enrolled Adult patients undergoing craniofacial surgery will be also selected and enrolled for craniofacial trauma and tumors To this purpose cranial bone tissue specimens derived from both paediatric and adult patients will be obtained as surgical waste during craniectomy or cranial vault remodeling upon obtaining the signed informed consent by parents in case of children For each patient the waste tissues will be randomized into three aliquots Of these 1 aliquot will be collected in cell growth medium for seeding bone tissue samples into culture plate to isolate mesenchymal stromal cells MSCs Thereafter MSC will be expanded up to the 3rd culture passage and collected in biobanking infrastructures with associated anonymized clinical data and optimized tracking system for subsequent analysis as follows GelMA-based bioink NP release and biocompatibility will be analyzed using MSC through functional assays to evaluate focal adhesion dynamics and study cell adhesion to the gelified bioink by immunofluorescence assay Cell behaviors and viability will be measured in time course experiments using a live cell imaging system Incucyte Live Cells Analysis systems
In addition 2 aliquots of patients-derived bone tissue specimens will be snap-frozen in liquid nitrogen after submerging it in cryoprotective medium with protease inhibitors Proteins and metabolites will be isolated from these samples using in-house standardized protocol Extracted samples will be then digested using Filter-aided sample preparation FASP digestion protocol Then samples will be analysed by Liquid Chromatography Tandem Mass Spectrometry LC-MSMS Also metabolites will be assessed by LC-MSMS and will be separated by HPLC Then obtained results on proteins and metabolites will be analysed by integrated pathway analysis using IPA to achieve the multiomic profiling of each patient thus identifying druggable targets to be exploited in drug design Computational drug design tools including molecular docking and virtual screening will be employed to identify the lead compounds from drugchemical databases capable of interacting with the selected targets from the integrated omic profile datasets The dynamic behavior stability and binding interactions of target-ligand complexes will be studied through Molecular dynamics MD simulations to analyze binding motifs and hotspots MD-informed insights on molecular interactions will guide the design and optimization of novel compounds This will involve incorporating structural modifications with ligand-based approaches and performing virtual screening to enhance binding affinity selectivity and drug-likeness The biomolecules identified through these in silico studies will be then commercially purchased and encapsulated in PLGA NPs In this regard nanoprecipitation and single emulsion will be exploited for hydrophobic payloads while double-emulsion solvent evaporation technique w1ow2 will be employed for hydrophilic payloads After PLGA delivering selected compounds will be complexed into hydrogel GelMA matrix The functional trophic and pro-regenerative properties of the bioink compound delivering biomolecules will be assessed in MSC to test osteogenic properties - through ALP activity assay in vitro mineralization staining and marker gene expression analysis In addition human umbilical vein endothelial cells commercial line will be treated with GelMA-NPs-drug complex for evaluating the angiogenic properties by counting and sizing the capillary-like structures formed in vitro tube formation assay
In addition a small aliquot of peripheral venous blood 2-3 ml for pediatric patients and up to 7 ml for adult patients from routine clinical examinations will be collected PBMCs will be isolated from whole blood sample using Ficoll density gradient centrifugation technique Monocytemacrophage cells will be then separated through CD14 antibody-conjugated magnetic beads and cultured in vitro with factors ie M-CSF RANKL to form mature osteoclasts These will be used to evaluate the bio-resorbability of GelMA-NP compound Briefly osteoclasts will be seeded on the GelMA-NP nanopatterned surfaces and the expression of typical osteoclast-specific markers will be assessed through immunofluorescence analysis SEM and immunofluorescence assay will be used to visualize ultra-structurally the osteoclast-bioink interface and discriminate resorption two- and three-dimensionally
MSCosteoprogenitors and osteoclasts derived from patients will be also exploited to obtain 2D and 3D in vitro culture to mimic the biological microenviroment existing at the bone-implant boundary for the validation of the developed bioink complex The bioink components will be either bioprinted with cells ie cells delivered through an independent nozzle to form cell-laden models or printed on the growth surface of cell culture vessels and let jelify before cell seeding according to the aims of each test The release dynamics of NP from the GelMA cell viability and proliferation angiogenic and bone regenerative effects will be assessed as mentioned above
To test its antibacterial effects both the unloaded GelMA and the GelMA-NP will be printed on to petri dishes and let jelify Thereafter it will be incubated with the opportunistic bacterial strains E coli S aureus Streptococcus mutans Enterococcus faecalis and Pseudomonas aeruginosa in the appropriate culture broth overnight Bacterial adhesion and count will be assessed comparatively testing dose- and time-related effects Antibacterial activity of GelMA-NP over GelMA will be also supported by SEM imaging
To this aim biocompatible PLGA-Polyethylene glycol PEG NPs will be synthesized and functionalized with bis-sulfone for binding targeting moieties on the surface The synthesis involves PLGA activation PLGA-PEG conjugation bis-sulfone activation and PLGA-PEG-bis-sulfone conjugation The PLGAPEG ratio will be adjusted for the hydrophobicityhydrophilicity payload Bis-sulfone is used for selective and efficient PEGylation of proteins disulfide bond or to conjugate His-tagged proteinpeptides according to standardized methods to implement either specific cell targeting or antimicrobial agents High performance liquid chromatography HPLC will be used to validate the biosynthetic reaction Dynamic Light Scattering DLS Nanoparticle Tracking Analysis NTA and Scanning electron microscopy SEM will define the morphological and ultrastructural properties of the final construct Surface Plasmon Resonance will validate target recognition Then GelMA-NP hybrid compound assembly will be achieved through extrusion 3D printing according to standardized production pipelines The hydrogel and the NP suspension will be dispensed through separate nozzles in 2D and 3D patterns with 100-1000 nm feature sizes under pressure and temperature control Scalar NP concentrations will be used and then evaluated
NP integrity and release dynamics of these from GelMA will be studied by submerging the GelMA-NPs loaded with fluorescent compound with a bio-mimetic cell growth medium and quantifying the NPs released in the supernatant in time course experiments NP quantification will be performed using UV-Vis Spectroscopy Fluorescence Spectroscopy or particle counting such as NTA system NPs integrity will be studied with transmission electron microscopy TEM and DLS
Once biochemically characterized GelMA-NPs these will be used to deliver bioactive compounds identified through proteomics data as subsequently described
To validate the developed bio-ink in in vitro craniofacial disease models patients with craniofacial malformations undergoing cranial surgery will be selected and enrolled in the study Specifically paediatric patients affected by craniosynostosis and other inborn defects trauma or brain tumors undergoing cranial surgery will be enrolled Adult patients undergoing craniofacial surgery will be also selected and enrolled for craniofacial trauma and tumors To this purpose cranial bone tissue specimens derived from both paediatric and adult patients will be obtained as surgical waste during craniectomy or cranial vault remodeling upon obtaining the signed informed consent by parents in case of children For each patient the waste tissues will be randomized into three aliquots Of these 1 aliquot will be collected in cell growth medium for seeding bone tissue samples into culture plate to isolate mesenchymal stromal cells MSCs Thereafter MSC will be expanded up to the 3rd culture passage and collected in biobanking infrastructures with associated anonymized clinical data and optimized tracking system for subsequent analysis as follows GelMA-based bioink NP release and biocompatibility will be analyzed using MSC through functional assays to evaluate focal adhesion dynamics and study cell adhesion to the gelified bioink by immunofluorescence assay Cell behaviors and viability will be measured in time course experiments using a live cell imaging system Incucyte Live Cells Analysis systems
In addition 2 aliquots of patients-derived bone tissue specimens will be snap-frozen in liquid nitrogen after submerging it in cryoprotective medium with protease inhibitors Proteins and metabolites will be isolated from these samples using in-house standardized protocol Extracted samples will be then digested using Filter-aided sample preparation FASP digestion protocol Then samples will be analysed by Liquid Chromatography Tandem Mass Spectrometry LC-MSMS Also metabolites will be assessed by LC-MSMS and will be separated by HPLC Then obtained results on proteins and metabolites will be analysed by integrated pathway analysis using IPA to achieve the multiomic profiling of each patient thus identifying druggable targets to be exploited in drug design Computational drug design tools including molecular docking and virtual screening will be employed to identify the lead compounds from drugchemical databases capable of interacting with the selected targets from the integrated omic profile datasets The dynamic behavior stability and binding interactions of target-ligand complexes will be studied through Molecular dynamics MD simulations to analyze binding motifs and hotspots MD-informed insights on molecular interactions will guide the design and optimization of novel compounds This will involve incorporating structural modifications with ligand-based approaches and performing virtual screening to enhance binding affinity selectivity and drug-likeness The biomolecules identified through these in silico studies will be then commercially purchased and encapsulated in PLGA NPs In this regard nanoprecipitation and single emulsion will be exploited for hydrophobic payloads while double-emulsion solvent evaporation technique w1ow2 will be employed for hydrophilic payloads After PLGA delivering selected compounds will be complexed into hydrogel GelMA matrix The functional trophic and pro-regenerative properties of the bioink compound delivering biomolecules will be assessed in MSC to test osteogenic properties - through ALP activity assay in vitro mineralization staining and marker gene expression analysis In addition human umbilical vein endothelial cells commercial line will be treated with GelMA-NPs-drug complex for evaluating the angiogenic properties by counting and sizing the capillary-like structures formed in vitro tube formation assay
In addition a small aliquot of peripheral venous blood 2-3 ml for pediatric patients and up to 7 ml for adult patients from routine clinical examinations will be collected PBMCs will be isolated from whole blood sample using Ficoll density gradient centrifugation technique Monocytemacrophage cells will be then separated through CD14 antibody-conjugated magnetic beads and cultured in vitro with factors ie M-CSF RANKL to form mature osteoclasts These will be used to evaluate the bio-resorbability of GelMA-NP compound Briefly osteoclasts will be seeded on the GelMA-NP nanopatterned surfaces and the expression of typical osteoclast-specific markers will be assessed through immunofluorescence analysis SEM and immunofluorescence assay will be used to visualize ultra-structurally the osteoclast-bioink interface and discriminate resorption two- and three-dimensionally
MSCosteoprogenitors and osteoclasts derived from patients will be also exploited to obtain 2D and 3D in vitro culture to mimic the biological microenviroment existing at the bone-implant boundary for the validation of the developed bioink complex The bioink components will be either bioprinted with cells ie cells delivered through an independent nozzle to form cell-laden models or printed on the growth surface of cell culture vessels and let jelify before cell seeding according to the aims of each test The release dynamics of NP from the GelMA cell viability and proliferation angiogenic and bone regenerative effects will be assessed as mentioned above
To test its antibacterial effects both the unloaded GelMA and the GelMA-NP will be printed on to petri dishes and let jelify Thereafter it will be incubated with the opportunistic bacterial strains E coli S aureus Streptococcus mutans Enterococcus faecalis and Pseudomonas aeruginosa in the appropriate culture broth overnight Bacterial adhesion and count will be assessed comparatively testing dose- and time-related effects Antibacterial activity of GelMA-NP over GelMA will be also supported by SEM imaging
Study Oversight
Has Oversight DMC:
None
Is a FDA Regulated Drug?:
None
Is a FDA Regulated Device?:
None
Is an Unapproved Device?:
None
Is a PPSD?:
None
Is a US Export?:
None
Is an FDA AA801 Violation?:
None