Merck
  • Home
  • Search Results
  • High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

High-throughput bone and cartilage micropellet manufacture, followed by assembly of micropellets into biphasic osteochondral tissue.

Cell and tissue research (2015-05-01)
Betul Kul Babur, Kathryn Futrega, William B Lott, Travis Jacob Klein, Justin Cooper-White, Michael Robert Doran
ABSTRACT

Engineered biphasic osteochondral tissues may have utility in cartilage defect repair. As bone-marrow-derived mesenchymal stem/stromal cells (MSC) have the capacity to make both bone-like and cartilage-like tissues, they are an ideal cell population for use in the manufacture of osteochondral tissues. Effective differentiation of MSC to bone-like and cartilage-like tissues requires two unique medium formulations and this presents a challenge both in achieving initial MSC differentiation and in maintaining tissue stability when the unified osteochondral tissue is subsequently cultured in a single medium formulation. In this proof-of-principle study, we used an in-house fabricated microwell platform to manufacture thousands of micropellets formed from 166 MSC each. We then characterized the development of bone-like and cartilage-like tissue formation in the micropellets maintained for 8-14 days in sequential combinations of osteogenic or chondrogenic induction medium. When bone-like or cartilage-like micropellets were induced for only 8 days, they displayed significant phenotypic changes when the osteogenic or chondrogenic induction medium, respectively, was swapped. Based on these data, we developed an extended 14-day protocol for the pre-culture of bone-like and cartilage-like micropellets in their respective induction medium. Unified osteochondral tissues were formed by layering 12,000 osteogenic micropellets and 12,000 chondrogenic micropellets into a biphasic structure and then further culture in chondrogenic induction medium. The assembled tissue was cultured for a further 8 days and characterized via histology. The micropellets had amalgamated into a continuous structure with distinctive bone-like and cartilage-like regions. This proof-of-concept study demonstrates the feasibility of micropellet assembly for the formation of osteochondral-like tissues for possible use in osteochondral defect repair.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
Sodium phosphate dibasic, BioXtra, ≥99.0%
Sigma-Aldrich
Sodium phosphate dibasic, BioReagent, suitable for cell culture, suitable for insect cell culture, ≥99.0%
Sigma-Aldrich
Sodium phosphate dibasic, ReagentPlus®, ≥99.0%
Sigma-Aldrich
Sodium phosphate dibasic, for molecular biology, ≥98.5% (titration)
Sigma-Aldrich
Dexamethasone, powder, BioReagent, suitable for cell culture, ≥97%
Sigma-Aldrich
Dexamethasone, meets USP testing specifications
Sigma-Aldrich
Calcium chloride, anhydrous, BioReagent, suitable for insect cell culture, suitable for plant cell culture, ≥96.0%
Sigma-Aldrich
Calcium chloride, meets USP testing specifications
Sigma-Aldrich
DAPI, for nucleic acid staining
Sigma-Aldrich
Methylene blue, certified by the Biological Stain Commission
Sigma-Aldrich
Calcium chloride, AnhydroBeads, −10 mesh, ≥99.99% trace metals basis
Sigma-Aldrich
Methylene Blue solution, 0.05 wt. % in H2O
Sigma-Aldrich
Dexamethasone, powder, γ-irradiated, BioXtra, suitable for cell culture, ≥80% (HPLC)
Sigma-Aldrich
Triton X-100, laboratory grade
Sigma-Aldrich
Dexamethasone, ≥98% (HPLC), powder
Sigma-Aldrich
o-Cresolphthalein Complexone, powder
Sigma-Aldrich
8-Hydroxyquinoline, ACS reagent, 99%
Sigma-Aldrich
Ethyl alcohol, Pure, 190 proof, ACS spectrophotometric grade, 95.0%
Sigma-Aldrich
Ethyl alcohol, Pure, 200 proof, anhydrous, ≥99.5%
Sigma-Aldrich
Calcium chloride, anhydrous, powder, 99.99% trace metals basis
Sigma-Aldrich
Calcium chloride, AnhydroBeads, −10 mesh, ≥99.9% trace metals basis
Sigma-Aldrich
Sodium phosphate dibasic, 99.95% trace metals basis
Sigma-Aldrich
Sodium phosphate dibasic, BioUltra, for molecular biology, ≥99.5% (T)
Sigma-Aldrich
Calcium chloride solution, BioUltra, for molecular biology, ~1 M in H2O
Sigma-Aldrich
Methylene Blue solution, for microscopy, concentrate according to Ehrlich, concentrated, aqueous solution
Sigma-Aldrich
5α-Androstan-17β-ol-3-one, purum, ≥99.0% (TLC)
Sigma-Aldrich
Thrombopoietin human, recombinant, expressed in E. coli, lyophilized powder, suitable for cell culture, ≥98% (SDS-PAGE and HPLC)
Sigma-Aldrich
8-Hydroxyquinoline, crystalline
Sigma-Aldrich
Calcium chloride, Vetec, reagent grade, 96%
Sigma-Aldrich
Sodium phosphate dibasic, Vetec, reagent grade, 99%