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Marrow stromal fibroblastic cell cultivation in vitro on decellularized bone marrow extracellular matrix
Timothy F Dutra1, Samuel W French
1St. Louis City Medical Examiner's Office, 1300 Clark Avenue, St. Louis, MO 63103, USA. tdmdphd@yahoo.com
Abstract:
The in vitro biocompatibility of decellularized bone marrow extracellular matrix was evaluated. Following a freeze-thaw cycle, sectioned discs of fresh frozen rat metaphyseal bone were sequentially incubated in solutions of hypertonic, then hypotonic Ringer's solution, followed by deoxycholic acid, then DNAase I. The adequacy of decellularization of marrow stroma was examined by light microscopy. Marrow stromal fibroblastic cells were harvested by dispersion of rat long bone marrow, followed by concentration by discontinuous Ficoll-Paque gradient centrifugation. The fibroblastic cells were expanded by in vitro cultivation, and second passage cells were cryopreserved until needed. Cryopreserved marrow stromal cells were applied dropwise to sections of decellularized bone marrow extracellular matrix, and cultured in BJGb medium with 20% fetal bovine serum for ten days. Mature cultures were formalin fixed, decalcified, and embedded in paraffin. Light microscopy of hematoxylin and eosin stained sections showed individual spindle cells invading the upper portion of the decellularized extracellular matrix, and also a monolayer of spindle cells on the upper surfaces of exposed trabecular and cortical bone. This experiment showed that decellularized marrow extracellular matrix is a biocompatible three dimensional in vitro substrate for marrow stromal fibroblastic cells.
Insights
Decellularized bone marrow extracellular matrix provides a biocompatible scaffold for marrow stromal cells in vitro. This 3D matrix supports cell invasion and growth, showing promise for tissue engineering applications.
Area of Science:
- Biomaterials Science
- Tissue Engineering
- Cell Biology
Background:
- The bone marrow extracellular matrix (BME C) is a complex biological material.
- Decellularization techniques aim to remove cellular components while preserving the ECM structure.
- Understanding the biocompatibility of decellularized ECM (dECM) is crucial for regenerative medicine.
Purpose of the Study:
- To evaluate the in vitro biocompatibility of decellularized bone marrow extracellular matrix (d BME C).
- To assess the ability of marrow stromal fibroblastic cells to infiltrate and grow on the d BME C scaffold.
Main Methods:
- Bone marrow extracellular matrix was decellularized using a multi-step process including freeze-thaw, Ringer's solutions, deoxycholic acid, and DNAase I.
- Marrow stromal fibroblastic cells were isolated, expanded in vitro, and cryopreserved.
- Cells were cultured on d BME C sections for ten days.
- Cultures were analyzed using light microscopy after H&E staining.
Main Results:
- Light microscopy confirmed successful decellularization of the marrow stroma.
- Marrow stromal fibroblastic cells demonstrated invasion into the upper regions of the d BME C.
- A monolayer of cells formed on the exposed bone surfaces within the scaffold.
- Cells exhibited spindle-like morphology consistent with fibroblastic cells.
Conclusions:
- Decellularized bone marrow extracellular matrix serves as a biocompatible three-dimensional in vitro substrate.
- The d BME C supports the infiltration and growth of marrow stromal fibroblastic cells.
- This finding supports the potential use of d BME C in bone marrow-related tissue engineering strategies.
