Clin Orthop Surg.  2014 Dec;6(4):455-461. 10.4055/cios.2014.6.4.455.

The Effect of Poloxamer 407-Based Hydrogel on the Osteoinductivity of Demineralized Bone Matrix

• Lee JH
• Baek HR
• Lee KM
• Lee HK
• Im SB
• Kim YS
• Lee JH
• Chang BS
• Lee CK

• KMID: 1794735
• DOI: http://doi.org/10.4055/cios.2014.6.4.455

Abstract

BACKGROUND
Demineralized bone matrix (DBM) is used for bone healing due to its osteoinductivity, but it requires a carrier for clinical application. Here, we report the effects on the osteoinductivity of DBM by use of a poloxamer 407-based hydrogel as the carrier, compared to sterile water.
METHODS
DBM-W and DBM-H represent 27 wt% of DBM with sterile water and DBM with a poloxamer 407-based hydrogel, respectively. Both of the compositions were applied to human mesenchymal stem cell (MSC) cultures, and monitored for alkaline phosphatase (ALP) staining and ALP activity. Six 10-week-old athymic nude rats were used for abdominal muscle grafting with either DBM-W or DBM-H, and were tested by plane radiography, microfocus X-ray computed tomography (CT), and decalcified histology to evaluate ectopic bone formation.
RESULTS
The DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, and significantly higher ALP activity at 7 and 14 days of treatment, compared to the DBM-H group. Plane radiography could not confirm the radio-opaque lesions in the rat ectopic bone formulation model. However, ectopic bone formation was observed in both groups by micro-CT. Compared to the DBM-H group, the DBM-W group showed higher bone volume, percent bone volume and trabecular number, and the difference in percent bone volume was statistically significant. Decalcified histology found bony tissue with lamellation in both groups.
CONCLUSIONS
Our results suggest that poloxamer 407-based hydrogel has efficacy as a DBM carrier since it shows ectopic bone formation, but its effects on the quality and quantity of osteoblastic differentiation in rat abdominal ectopic bone and MSC are considered negative.

Keyword

Demineralized bone matrix; Osteoinductivity; Carrier; Poloxamer 407-based hydrogel

MeSH Terms

Animals
Bone Matrix/*physiology
Cell Culture Techniques
Decalcification Technique
Excipients/*pharmacology
Hydrogels/pharmacology
Male
Mesenchymal Stromal Cells/*drug effects
Osteogenesis/*drug effects
Poloxamer/*pharmacology
Rats
Rats, Nude
Excipients
Hydrogels
Poloxamer

Figure

• 

  Fig. 1 Operation process. Three incisions and pouches were made per side (right and left) at the abdominal muscle of athymic nude rats. (A) A picture of fixed demineralized bone matrix (DBM). (B) A picture of the sutured pouches after DBM implantation.

• 

  Fig. 2 Alkaline phosphatase (ALP) staining and ALP activity assay. (A) DBM-W group showed stronger ALP staining at 7, 14, and 21 days of treatment, although the staining faded in both groups at 21 days. (B) ALP activity was significantly higher in the DBM-W group at 7 and 14 days of treatment (p < 0.001 for DBM-W; p = 0.0003 for DBM-H). Although 21 days of treatment showed the same pattern, no statistical significance was found (p = 0.166). DBM: demineralized bone matrix, DBM-W: DBM with sterile water, DBM-H: DBM with poloxamer 407-based hydrogel, D: day.

• 

  Fig. 3 Anteroposterior abdominal radiographs obtained from DBM-W group (A) and DBM-H group (B) after 8 weeks of implantation. Easily identifiable radio-opaque lesions were not observed in either group. DBM: demineralized bone matrix, DBM-W: DBM with sterile water, DBM-H: DBM with poloxamer 407-based hydrogel.

• 

  Fig. 4 Microfocus X-ray computed tomography of a specimen from the DBM-W group (A) and DBM-H group (B) after 8 weeks of implantation. For both A and B, each panel represents a transaxial image, sagittal image, and coronal image (from left to right). DBM: demineralized bone matrix, DBM-W: DBM with sterile water, DBM-H: DBM with poloxamer 407-based hydrogel.

• 

  Fig. 5 Decalcified histology of the rats in the DBM-W group (A) and DBM-H group (B) after 8 weeks of implantation (H&E, × 12.5 and × 100). Under low magnification, both of the groups showed newly formed bone surrounded by abdominal muscle. Under higher magnification, the lamellate tissue and lacunar space were arranged concentrically, and a haversian canal was observed in the center, confirming bone remodeling. DBM: demineralized bone matrix, DBM-W: DBM with sterile water, DBM-H: DBM with poloxamer 407-based hydrogel, arrow heads: pre-existing abdominal muscle, arrows: newly formed ectopic calcified tissue.

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