title: Processing large-diameter poly(L-lactic acid) microfiber mesh/mesenchymal stromal cell constructs via resin embedding: an efficient histologic method.
abstract: In this study, we performed a complete histologic analysis of constructs based on large diameter ( >100 μm) poly-L-lactic acid (PLLA) microfibers obtained via dry-wet spinning and rat Mesenchymal Stromal Cells (rMSCs) differentiated towards the osteogenic lineage, using acrylic resin embedding. In many synthetic polymer-based microfiber meshes, ex post processability of fiber/cell constructs for histologic analysis may face deterring difficulties, leading to an incomplete investigation of the potential of these scaffolds. Indeed, while polymeric nanofiber (fiber diameter = tens of nanometers)/cell constructs can usually be embedded in common histologic media and easily sectioned, preserving the material structure and the antigenic reactivity, histologic analysis of large polymeric microfiber/cell constructs in the literature is really scant. This affects microfiber scaffolds based on FDA-approved and widely used polymers such as PLLA and its copolymers. Indeed, for such constructs, especially those with fiber diameter and fiber interspace much larger than cell size, standard histologic processing is usually inefficient due to inhomogeneous hardness and lack of cohesion between the synthetic and the biological phases under sectioning. In this study, the microfiber/MSC constructs were embedded in acrylic resin and the staining/reaction procedures were calibrated to demonstrate the possibility of successfully employing histologic methods in tissue engineering studies even in such difficult cases. We histologically investigated the main osteogenic markers and extracellular matrix molecules, such as alkaline phosphatase, osteopontin, osteocalcin, TGF-β1, Runx2, Collagen type I and the presence of amorphous, fibrillar and mineralized matrix. Biochemical tests were employed to confirm our findings. This protocol permitted efficient sectioning of the treated constructs and good penetration of the histologic reagents, thus allowing distribution and expression of almost all the tested molecules to be revealed. Our results demonstrated that it is possible to perform histologic analyses of large-diameter PLLA-based microfiber scaffold/MSC constructs that face the failure of standard histologic procedures.
authors.affiliation: Center for Clinical Use of Stem Cells (CUCCS-RRMR), University of Pisa, 56126 Pisa, Italy. Department of Surgical, Medical, Molecular Pathology and Emergency Medicine, University of Pisa, 56124 Pisa, Italy.
authors.forename: Delfo
authors.initials: D
authors.lastname: D'Alessandro
authors.forename: Gianni
authors.initials: G
authors.lastname: Pertici
authors.forename: Stefania
authors.initials: S
authors.lastname: Moscato
authors.forename: Maria Rita
authors.initials: MR
authors.lastname: Metelli
authors.forename: Sabrina
authors.initials: S
authors.lastname: Danti
authors.forename: Claudia
authors.initials: C
authors.lastname: Nesti
authors.forename: Stefano
authors.initials: S
authors.lastname: Berrettini
authors.forename: Mario
authors.initials: M
authors.lastname: Petrini
authors.forename: Serena
authors.initials: S
authors.lastname: Danti
mesh_terms: D000180:Acrylic Resins / Q000737:chemistry*; D000423:Alcian Blue / Q000737:chemistry; D000469:Alkaline Phosphatase / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000737:chemistry; D019070:Cell Lineage; D002470:Cell Survival; D004247:DNA / Q000032:analysis; D005109:Extracellular Matrix / Q000378:metabolism; D005269:Femur / Q000473:pathology; D006652:Histological Techniques; D006801:Humans; D019344:Lactic Acid / Q000737:chemistry*; D059630:Mesenchymal Stem Cells / Q000166:cytology*; D010012:Osteogenesis; D010503:Periodic Acid-Schiff Reaction; D011091:Polyesters; D011108:Polymers / Q000737:chemistry*; D051381:Rats; D017208:Rats, Wistar; D013977:Tibia / Q000473:pathology; D014048:Tolonium Chloride / Q000737:chemistry
chemical_list: D000180:Acrylic Resins; D011091:Polyesters; D011108:Polymers; D014048:Tolonium Chloride; D019344:Lactic Acid; C033616:poly(lactide); D004247:DNA; D000469:Alkaline Phosphatase; D000423:Alcian Blue; D002118:Calcium