Effect of porous polycaprolactone beads on bone regeneration: preliminary in vitro and in vivostudies
© Byun et al.; licensee BioMed Central Ltd. 2014
Received: 18 July 2014
Accepted: 19 October 2014
Published: 24 November 2014
For the effective bone regeneration with appropriate pathological/physiological properties, a variety of bone fillers have been adapted as a therapeutic treatment. However, the development of ideal bone fillers is still remained as a big challenge in clinical practice. The main aims of this study are i) fabrication of a highly porous PCL beads; and ii) the estimation of the potential use of the porous PCL beads as a bone filler through preliminary animal study.
The porous PCL beads with size range of 53 ~ 600 μm (425 ~ 500 μm dominantly) are fabricated by a spray/precipitation method using a double nozzle spray and PCL solution (in tetraglycol). The PCL beads show highly porous inner pore structure and the pores are interconnected with outer surface pores. For the preliminary animal study, we recognize that the porous PCL bead can induce the new bone formation from the outer surface of bone defect toward the bone marrow cavity through the bead matrix.
From the preliminary results, we can suggest that the highly porous PCL beads may be a promising candidate as a bone filler (scaffolding matrix) for the effective bone regeneration.
Although an injury bone can be reconstructed spontaneously, large bone defects created by trauma, tumor resection, corrective osteotomy, and congenital deformity are considered as a notable challenge for orthopedic and oral/maxiallofacial surgeons . For the effective bone regeneration with appropriate pathological/physiological properties, a variety of bone grafts including biological and synthetic biomaterials have been utilized as a therapeutic treatment. The biological grafts (autograft and allograft) are commonly used as a first line therapy for large-sized bone defect, but insufficient donor materials, inevitable donor site morbidity, risk of infection (autograft); and risk of immune response/disease transmission (allograft) remain as significant limitations in the clinical practice [2–4]. To solve the limitations, ceramic-based materials with similar mineral constituent of bone, such as hydroxyapatite (HA) and tri-calcium phosphate (TCP) have been utilized for the effective bone regeneration due to their biocompatibility, non-immunogenecity, osteoconductivity, bonding affinity with host bone, etc. [4–8]. However, their low reliability (i.e., weak mechanical strengths and high fragile failure rate) in wet environment which leads to difficulty for load-bearing applications and long-term degradation rate which can prohibit new bone growth into the defect site are considered as a limitation for clinical applications [5, 9, 10]. Recently, US Food and Drug Administration (FDA) approved biodegradable polymers [e.g., poly(glycolic acid) (PGA), poly(lactic acid) (PLA) and poly(lactic acid-co-glycolic acid) (PLGA), poly(ϵ-caprolactone) (PCL), polydioxanone (PDO)] with biocompatibility, predictable degradation rate and controllable mechanical properties are gained increasing interest as alternative matrices for bone regeneration . Among them, the PCL is considered as a more promising matrix for bone regeneration compared to the other biodegradable polymers because of its no acidic by-products formation during degradation, flexibility (vs. PGA, PLA, PLGA); and relatively long-term structural stability which can provide a frame work during bone regeneration (vs. PGA, PLGA, PDO). Low et al.  demonstrated that the PCL matrix can allow biomimetic environment for the initial blood coagulation, cell infiltration, new blood vessel formation, and effective long-term osteogenesis. Moreover, Schantz et al.  reported that the PCL matrix is well tolerated in vivo and integrated with the host bone, suggesting that the PCL matrix may be a suitable graft for bone regeneration. Nevertheless the encouraging results, the use of PCL matrix as a bone filler is still limited, probably due to the concern about long-term remaining at applied site of dense PCL matrix which may prevent new bone formation. However, we expected that the highly porous PCL matrix may allow an appropriate environment for initial bone growth (by structural stability), accelerated degradation (by large surface area), sustained delivery of bioactive molecules (by high porosity), and thus become a good candidate as a bone filler.
Therefore, the main aims of this study are i) fabrication of a highly porous PCL bead; and ii) the estimation of the potential use of the porous PCL bead as a bone filler through preliminary animal study. To achieve this goal, porous PCL beads are fabricated by a spray/precipitation method using a double nozzle spray and PCL solution (in tetraglycol). The tetraglycol which is frequently utilized in parenteral delivery [14–16] is used as a nontoxic solvent for PCL. The preliminary animal study (femur defect rat model) to estimate the bone regeneration behavior by the porous PCL bead is also investigated.
PCL (Mw 80,000 Da) and tetraglycol (glycofurol) as a nontoxic solvent for PCL were purchased from Sigma-Aldrich (USA). All other chemicals were analytical grade and were used as received. Ultrapure grade water (>18 mΩ) was purified using a Milli-Q purification system (Millipore Co., USA). For animal study, the porous PCL beads were sterilized by ethylene oxide (EO).
Preparation of porous PCL beads
Characterization of porous PCL beads
Morphology observation and porosity measurement
The morphology of prepared porous PCL beads was observed by a field emission scanning electron microscope (FE-SEM; Model S-4300, Hitachi, Japan). The cross-sectional specimen was prepared by cutting them using a blade after being frozen in liquid nitrogen. The porosity of the PCL beads was estimated using mercury porosimetry (Poresizer 9320; Micromeritics, USA). To determine the porosity, it was assumed that the surface tension of mercury is 480 dyne/cm .
Preliminary animal study
Results and discussion
Preparation and characterization of porous PCL beads
It was observed that the size range of the prepared porous PCL beads in our procedure [purging rate of N2, 2.5 L/min (outer nozzle) and feeding rate of PCL solution, 60 mL/h (inner nozzle)] is 53 ~ 600 μm and the porous PCL beads with size range of 425 ~ 500 μm is more dominant than other size ranges. Their size distribution also can be controlled by the purging rate of N2 and/or feeding rate of polymer solution [higher N2 purging rate, smaller size dominantly (lower volume ratio of polymer solution to N2 gas); higher PCL solution feeding rate, larger size dominantly (higher volume ratio of polymer solution to N2 gas); not shown data), as expected. The porous PCL beads exhibited highly porous inner pore structures and the pores are interconnected with surface pores. The formation of porous structure can be understood by phase separation between polymer (PCL) solution and nonsolvent [solvent (tetraglycol)-nonsolvent (50% ethanol) exchange] during the precipitation of the PCL solution in coagulation bath . From the morphology results, we could expect that the highly porous structure can provide large surface area of PCL matrix and thus may accelerate the degradation rate which can allow appropriate space for new bone formation, moreover the porous beads may be a reservoir of bioactive molecules for bone regeneration (e.g., drugs, growth factors, cytokines, etc.). Therefore, we believed that the porous PCL beads may be a promising matrix for effective bone regeneration. The porosity of the porous PCL beads measured using mercury porosimetry were over 80%, regardless of bead size. The porous PCL beads with size range of 425 ~ 500 μm were selected for the preliminary animal study using rat model .
Preliminary animal study
We prepared porous PCL beads by a spray/precipitation method using a double nozzle spray and PCL solution (in tetraglycol). It was observed that the size range of prepared porous PCL beads (purging rate of N2, 2.5 L/min and feeding rate of PCL solution, 60 mL/hr) is 53 ~ 600 μm (dominant size range, 425 ~ 500 μm) and their size distribution can be controlled by the purging rate of N2 and/or feeding rate of polymer solution. The porous PCL beads showed highly porous inner pore structure and the pores are interconnected with outer surface pores. For the preliminary animal study, we recognized that the porous PCL bead can induce the new bone formation from the outer surface of bone defect toward the bone marrow cavity through the bead matrix. From the preliminary results, we could suggest that the highly porous PCL beads may be a promising candidate as a matrix for the bone regeneration. The long-term studies (i.e., in vivo degradation rate of the porous PCL beads and bone regeneration/maturation behaviors through the matrix) using a large animal (porcine) to confirm the potential use of the porous PCL beads as a clinically applicable bone filler are in progress.
Availability of supporting data
The data sets supporting the results of this article are included within the article.
This research was supported by a grant of the Korea Health Technology R&D Project through the Korea Health Industry Development Institute (KHIDI), funded by the Ministry of Health & Welfare, Republic of Korea (grant number: HI13C1596).
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