Regulating in vivo calcification of alginate microbeads

Abstract

Alginate calcification has been previously reported clinically and during animal implantation; however no study has investigated the mechanism, extensively characterized the mineral, or evaluated multiple methods to regulate or eliminate mineralization. In the present study, alginate calcification was first studied in vitro: calcium-crosslinked alginate beads sequestered surrounding phosphate while forming traces of hydroxyapatite. Calcification in vivo was then examined in nude mice using alginate microbeads with and without adipose stem cells (ASCs). Variables included the delivery method, site of delivery, sex of the animal, time in vivo, crosslinking solution, and method of storage prior to delivery. Calcium-crosslinked alginate microbeads mineralized when injected subcutaneously or implanted intramuscularly after 1–6 months. More extensive analysis with histology, microCT, FTIR, XRD, and EDS showed calcium phosphate deposits throughout the microbeads with surface mineralization that closely matched hydroxyapatite found in bone. Incorporating 25 mM bisphosphonate reduced alginate calcification whereas using barium chloride eliminated mineralization. Buffering the crosslinking solution with HEPES at pH 7.3 while washing and storing samples in basal media prior to implantation also eliminated calcification in vivo. This study shows that alginate processing prior to implantation can significantly influence bulk hydroxyapatite formation and presents a method to regulate alginate calcification.

Bone Regeneration Potential of Stem Cells Derived from Periodontal Ligament or Gingival Tissue Sources Encapsulated in RGD-Modified Alginate Scaffold

“…Further, it has been reported that presentation of RGD tripeptide to alginate scaffold can promote osteoblast proliferation, leading to increased bone regeneration capacity. 24,28 In our current in vitro study, we confirmed that RGD-modified alginate microspheres containing dental MSCs indeed produced significantly more mineralized tissue than was generated by unmodified alginate hydrogel.…”

“…25,26 Due to their unique properties including gentle gelation behavior, biodegradability, biocompatibility, easy cell encapsulation/cell recovery, and chemical versatility, they have a wide variety of biomedical applications, such as the encapsulation of cells and sensitive bioactive molecules to facilitate minimally invasive surgical procedures. [27][28][29] In our previous studies, we have utilized alginate hydrogels as a scaffold for the encapsulation of PDLSCs and GMSCs. 19,20 We showed that an alginate encapsulation system has the potential to enhance hard tissue regeneration in vitro and in vivo, making it a promising candidate for minimally invasive dental and orthopedic applications.…”

The in vitro and in vivo capacity of culture-expanded human cells from several sources encapsulated in alginate to form cartilage

“…To our knowledge, calcium-cross-linked alginate calcifies through binding the surrounding phosphate ions to form calcium phosphate crystals. These crystals are stable in neutral to basic environments and do not appear at pH of less than 6.8 (Lee et al, 2010). We believe that the calcified constructs were possibly generated in a neutral to alkaline environment prior to implantation, since these constructs were either not metabolically active (non-seeded alginate) or had a low metabolic activity due to stable cartilage formation (ACs) or due to the deficiency of TGFβ1.…”

Nanostructured Injectable Cell Microcarriers for Tissue Regeneration

“…In addition, alginate has been used for bone regeneration, considering its calcification capacity in vivo. This results from interactions between Ca 2+ in the cross-linked alginate and the surrounding phosphate ions [101], making it a useful base material for orthopedic applications. However, the slow and uncontrolled degradation in vivo and poor cell adhesion properties of alginate must be addressed [102].…”