title: Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.
abstract: Bone tissue engineering via endochondral ossification has been explored by chondrogenically priming cells using soluble mediators for at least 3 weeks to produce a hypertrophic cartilage template. Although recapitulation of endochondral ossification has been achieved, long-term in vitro culture is required for priming cells through repeated supplementation of inductive factors in the media. To address this challenge, a microparticle-based growth factor delivery system was engineered to drive endochondral ossification within human bone marrow-derived mesenchymal stem cell (hMSC) aggregates. Sequential exogenous presentation of soluble transforming growth factor-β1 (TGF-β1) and bone morphogenetic protein-2 (BMP-2) at various defined time courses resulted in varying degrees of chondrogenesis and osteogenesis as demonstrated by glycosaminoglycan and calcium content. The time course that best induced endochondral ossification was used to guide the development of the microparticle-based controlled delivery system for TGF-β1 and BMP-2. Gelatin microparticles capable of relatively rapid release of TGF-β1 and mineral-coated hydroxyapatite microparticles permitting more sustained release of BMP-2 were then incorporated within hMSC aggregates and cultured for 5 weeks following the predetermined time course for sequential presentation of bioactive signals. Compared with cell-only aggregates treated with exogenous growth factors, aggregates with incorporated TGF-β1- and BMP-2-loaded microparticles exhibited enhanced chondrogenesis and alkaline phosphatase activity at week 2 and a greater degree of mineralization by week 5. Staining for types I and II collagen, osteopontin, and osteocalcin revealed the presence of cartilage and bone. This microparticle-incorporated system has potential as a readily implantable therapy for healing bone defects without the need for long-term in vitro chondrogenic priming. Significance: This study demonstrates the regulation of chondrogenesis and osteogenesis with regard to endochondral bone formation in high-density stem cell systems through the controlled presentation of inductive factors from incorporated microparticles. This work lays the foundation for a rapidly implantable tissue engineering system that promotes bone repair via endochondral ossification, a pathway that can delay the need for a functional vascular network and has an intrinsic ability to promote angiogenesis. The modular nature of this system lends well to using different cell types and/or growth factors to induce endochondral bone formation, as well as the production of other tissue types.
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Phuong N
authors.initials: PN
authors.lastname: Dang
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Neha
authors.initials: N
authors.lastname: Dwivedi
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Lauren M
authors.initials: LM
authors.lastname: Phillips
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA.
authors.forename: Xiaohua
authors.initials: X
authors.lastname: Yu
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Samuel
authors.initials: S
authors.lastname: Herberg
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Caitlin
authors.initials: C
authors.lastname: Bowerman
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA.
authors.forename: Loran D
authors.initials: LD
authors.lastname: Solorio
authors.affiliation: Department of Biomedical Engineering, University of Wisconsin, Madison, Wisconsin, USA Department of Orthopedics and Rehabilitation, University of Wisconsin, Madison, Wisconsin, USA.
authors.forename: William L
authors.initials: WL
authors.lastname: Murphy
authors.affiliation: Department of Biomedical Engineering, Case Western Reserve University, Cleveland, Ohio, USA Department of Orthopaedic Surgery, Case Western Reserve University, Cleveland, Ohio, USA eben.alsberg@case.edu.
authors.forename: Eben
authors.initials: E
authors.lastname: Alsberg
mesh_terms: D000469:Alkaline Phosphatase / Q000235:genetics / Q000378:metabolism; D015415:Biomarkers / Q000378:metabolism; D001854:Bone Marrow Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D055396:Bone Morphogenetic Protein 2 / Q000378:metabolism / Q000494:pharmacology*; D002113:Calcification, Physiologic / Q000187:drug effects / Q000235:genetics; D002118:Calcium / Q000378:metabolism; D002449:Cell Aggregation; D020219:Chondrogenesis / Q000187:drug effects* / Q000235:genetics; D024042:Collagen Type I / Q000235:genetics / Q000378:metabolism; D024043:Collagen Type II / Q000235:genetics / Q000378:metabolism; D003692:Delayed-Action Preparations; D004339:Drug Compounding; D017886:Durapatite / Q000737:chemistry; D005780:Gelatin / Q000737:chemistry; D015870:Gene Expression; D006025:Glycosaminoglycans / Q000378:metabolism; D006801:Humans; D059630:Mesenchymal Stem Cells / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D015675:Osteocalcin / Q000235:genetics / Q000378:metabolism; D010012:Osteogenesis / Q000187:drug effects* / Q000235:genetics; D053495:Osteopontin / Q000235:genetics / Q000378:metabolism; D061251:Primary Cell Culture; D023822:Tissue Engineering / Q000379:methods; D053773:Transforming Growth Factor beta1 / Q000378:metabolism / Q000494:pharmacology*
chemical_list: C525718:BMP2 protein, human; D015415:Biomarkers; D055396:Bone Morphogenetic Protein 2; D024042:Collagen Type I; D024043:Collagen Type II; D003692:Delayed-Action Preparations; D006025:Glycosaminoglycans; D053773:Transforming Growth Factor beta1; D015675:Osteocalcin; D053495:Osteopontin; D005780:Gelatin; D017886:Durapatite; D000469:Alkaline Phosphatase; D002118:Calcium
keywords: Adult human bone marrow; Bone; Growth factor delivery; High-density culture; Mesenchymal stem cells; Tissue regeneration