Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats

Abstract

Objectives

Calcium phosphate cement (CPC) is promising for dental and craniofacial applications due to its ability to be injected or filled into complex-shaped bone defects and molded for esthetics, and its resorbability and replacement by new bone. The objective of this study was to investigate bone regeneration via novel macroporous CPC containing absorbable fibers, hydrogel microbeads and growth factors in critical-sized cranial defects in rats.

Methods

Mannitol porogen and alginate hydrogel microbeads were incorporated into CPC. Absorbable fibers were used to provide mechanical reinforcement to CPC scaffolds. Six CPC groups were tested in rats: (1) control CPC without macropores and microbeads; (2) macroporous CPC + large fiber; (3) macroporous CPC + large fiber + nanofiber; (4) same as (3), but with rhBMP2 in CPC matrix; (5) same as (3), but with rhBMP2 in CPC matrix + rhTGF-β1 in microbeads; (6) same as (3), but with rhBMP2 in CPC matrix + VEGF in microbeads. Rats were sacrificed at 4 and 24 weeks for histological and micro-CT analyses.

Results

The macroporous CPC scaffolds containing porogen, absorbable fibers and hydrogel microbeads had mechanical properties similar to cancellous bone. At 4 weeks, the new bone area fraction (mean ± sd; n = 5) in CPC control group was the lowest at (14.8 ± 3.3)%, and that of group 6 (rhBMP2 + VEGF) was (31.0 ± 13.8)% ( p < 0.05). At 24 weeks, group 4 (rhBMP2) had the most new bone of (38.8 ± 15.6)%, higher than (12.7 ± 5.3)% of CPC control ( p < 0.05). Micro-CT revealed nearly complete bridging of the critical-sized defects with new bone for several macroporous CPC groups, compared to much less new bone formation for CPC control.

Significance

Macroporous CPC scaffolds containing porogen, fibers and microbeads with growth factors were investigated in rat cranial defects for the first time. Macroporous CPCs had new bone up to 2-fold that of traditional CPC control at 4 weeks, and 3-fold that of traditional CPC at 24 weeks, and hence may be useful for dental, craniofacial and orthopedic applications.

Introduction

Bone defects are a worldwide problem, and the need for bone repair arises due to trauma, tumor ablative surgery, congenital defects, infectious conditions and other causes of loss of skeletal tissue . Furthermore, the need for bone repair is increasing rapidly due to an aging population with increased life expectancies and diseases such as osteoporosis . In particular, the supply of bone grafts to treat critical-sized bone defects remains a major challenging health issue worldwide . Although autograft and allograft transplantations are used clinically, their disadvantages include donor site morbidity, harvesting limitation, and disease transmission risks. Biomaterials are a promising alternative for bone repair. While bio-inert implants can induce fibrous-capsules, bioactive implants with bone-like calcium phosphate (CaP) minerals can bond to bone to form a functional interface. CaP minerals enhance cell attachment and osteoblastic phenotype expression, and hence are important for bone repair .

For sintered bioceramics to fit into a bone cavity, the surgeon needs to machine the graft or carve the surgical site, leading to increases in bone loss, trauma, and surgical time. Calcium phosphate cements can be molded to achieve esthetics in craniofacial repairs, and set in situ to provide intimate adaptation to complex-shaped defects . One such cement is comprised of tetracalcium phosphate (TTCP) and dicalcium phosphate-anhydrous (DCPA) and is referred to as CPC . The CPC powder can be mixed with an aqueous liquid to form a paste that can be sculpted during surgery to conform to the defects in hard tissues. The paste self-hardens to form resorbable hydroxyapatite . Traditional CPC was mechanically weak, hence absorbable fibers and chitosan were used to reinforce the CPC scaffold . Chitosan enabled CPC to be fast-setting and washout-resistant . Macropores were created in CPC using water-soluble mannitol porogen to enhance cell infiltration . Recently, alginate microbeads were incorporated into CPC as a potential vehicle for growth factor/cell delivery, and the CPC paste containing microbeads and reinforcement fibers was fully injectable . Macroporous CPC scaffold is promising for a variety of dental and craniofacial applications, including mandibular and maxillary ridge augmentation and periodontal bone repair, since CPC could be molded to the desired shape and set to form a scaffold for bone in growth. Other applications include the major reconstructions of the maxilla or mandible after trauma or tumor resection, as well as the support of metal dental implants or augmentation of deficient implant sites, and the repair of cranial defects. However, previous studies on macroporous CPC focused on in vitro experiments , without testing in animal models.

The objective of this study was to investigate bone regeneration via macroporous CPC containing absorbable fibers, microbeads and growth factors in a critical-sized cranial defect model in rats. It was hypothesized that: (1) while macropores will weaken the CPC mechanically, fiber reinforcement will increase the strength of CPC; (2) new bone formation will be increased via the macropores in CPC, and the new bone area fraction in the cranial defect will be increased via the incorporation of recombinant human bone morphogenetic protein-2 (rhBMP2), vascular endothelial growth factor (VEGF) and recombinant human transforming growth factor-β1 (rhTGF-β1) incorporated into CPC scaffolds.

Materials and methods

CPC composite scaffold fabrication

TTCP (Ca 4 (PO 4 ) 2 O) was synthesized from a solid-state reaction at 1500 °C between DCPA (CaHPO 4 ) and CaCO 3 (J.T. Baker, Phillipsburg, NJ). The mixture was ground to obtain TTCP particles with sizes of 1–80 μm, with a median particle size of 17 μm. DCPA was ground to obtain particles with sizes of 0.4–3.0 μm, with a median particle size of 1.0 μm. The TTCP and DCPA were then mixed to form the CPC powder. Traditionally, the TTCP/DCPA molar ratio was 1/1 . Recently, a TTCP/DCPA ratio of 1/3 was shown to yield CPC with strength similar to that using 1/1, while the 1/3 ratio had faster dissolution which indicates potentially faster resorption in vivo . Hence, in this study, the CPC control used a TTCP/DCPA ratio of 1/1, while the other groups used a TTCP/DCPA ratio of 1/3. Hence the TTCP/DCPA ratio of the CPC control was the same as the CPC in previous in vivo studies . Previous studies incorporated water-soluble mannitol into CPC to create macropores , and alginate microbeads into CPC as drug and cell carriers . While macroporous CPC was mechanically-weaker than CPC without macropores, chitosan and fibers were incorporated into CPC for reinforcement. Therefore, for mechanical testing, four CPCs were made: (i) CPC control (without mannitol or microbeads); (ii) macroporous CPC with 30% mannitol, 30% alginate microbeads, and 15% chitosan in CPC liquid (referred to as “macroporous CPC”); (iii) same as (ii), but with 15% large-diameter absorbable fibers (“macroporous CPC + large fiber”); (iv) same as (ii), but with 7.5% large-diameter fibers + 7.5% nanofibers (“macroporous CPC + large fiber + nanofiber”).

Alginate hydrogel microbeads were synthesized using a previously-described method . First, the alginate was oxidized to increase its degradability in vivo . Oxidation was performed using sodium periodate at the correct stoichiometric ratio of sodium periodate/alginate to have certain percentages of alginate oxidation . The percentage of oxidation (%) was the number of oxidized uronate residues per 100 uronate units in the alginate chain. Alginate at 7.5% oxidation was synthesized as described previously . Briefly, 1% of sodium alginate was dissolved in water. Then 1.51 mL of 0.25 mol/L sodium periodate (Sigma–Aldrich) was added to 100 mL alginate solution. At 24 h, the oxidization reaction was stopped by adding 1 g of ethylene glycol and then 2.5 g of sodium chloride. The precipitates were re-dissolved and the second precipitates were collected. The oxidized alginate thus obtained was dissolved in saline at a concentration of 1.2%. The solution was loaded into a bead-generating device (Var-J1, Nisco, Zurich, Switzerland). The droplets fell into a well of 100 mmol/L calcium chloride and crosslinked to form microbeads. The microbeads were slightly elongated with a mean length of 335 μm and a mean diameter of 232 μm as measured previously .

A suture fiber (Vicryl, Ethicon, Somerville, NJ) was cut into 3 mm filaments. This suture consisted of fibers braided into a bundle with a diameter of 322 μm, suitable for producing long cylindrical macropores after fiber dissolution in CPC. Chitosan malate (Vanson, Redmond, WA) was mixed with water at a chitosan/(chitosan + water) mass fraction of 15% to form the CPC liquid. Nanofibers of poly( d , l -lactide- co -glycolide) (PLGA) at a poly(lactic acid) (PLA)/poly(glycolic acid) (PGA) ratio of 50/50 were electrospun into non-woven mats, following a previous study . The mat was cut into 3 × 3 mm squares, because a previous study showed that 3 mm fibers in CPC were injectable through a 10-gauge needle . Another study also cut electrospun fibers into 3 mm dimensions for use in CPC . CPC powder and liquid were mixed at a mass ratio of 2:1, and the prescribed amounts of fibers and microbeads were mixed with the CPC–chitosan paste. The mixed composite paste was placed into 3 mm × 4 mm × 25 mm molds and set at 100% humidity for 4 h at 37 °C. The specimens were then demolded and immersed in water at 37 °C for 20 h.

Mechanical testing

A three-point flexural test was performed using a 20 mm span and a displacement rate of 1 mm/min to measure the mechanical properties of CPC. Flexural strength was calculated as S = 3 F max L /(2 bh 2 ), where F max is the maximum load, L is the span, b is specimen width, and h is specimen thickness. Elastic modulus was calculated as E = ( F / c )( L 3 /[4 bh 3 ]), where load F divided by displacement c is the slope of the load–displacement curve in the linear elastic region. Work-of-fracture (toughness) was measured as the area of the load–displacement curve divided by the specimen’s cross-sectional area .

Critical-sized cranial defect model in rats

The mechanical property results showed that the macroporous CPC group (ii) was much weaker than CPC control (i). However, fiber reinforcement rendered the macroporous CPC (iii and iv) to have similar strengths to CPC control. Therefore, for the animal study, the following six groups were tested:

  • (1)

    Control CPC without macropores and microbeads (similar to the CPC in previous in vivo studies, such as Friedman et al. );

  • (2)

    Macroporous CPC + large fiber (30% mannitol, 30% alginate microbeads, 15% chitosan in cement liquid, reinforced with 15% large-diameter Vicryl fibers);

  • (3)

    Macroporous CPC + large fiber + nanofiber (same as 2, except reinforcement with 7.5% Vicryl fibers + 7.5% nanofibers);

  • (4)

    Same as (3), but with rhBMP2 in the CPC matrix;

  • (5)

    Same as (3), but with rhBMP2 in the CPC matrix + rhTGF-β1 in the microbeads;

  • (6)

    Same as (3), but with rhBMP2 in the CPC matrix + VEGF in the microbeads.

rhBMP2, VEGF and rhTGF-β1 were commercially-obtained (PeproTech, Rocky Hill, NJ). Their amounts were 10 μg rhBMP2/defect, 6 μg VEGF/defect, and 200 ng rhTGF-β1/defect, selected following previous studies .

The rat critical-sized cranial defect model was approved by Harvard University (# 24-09) and University of Maryland (# 0909014). NIH procedures were followed. Surgery was performed at Harvard University. Lewis rats (age: 49–55 days, Charles River, Wilmington, MA) were anesthetized by intraperitoneal injection (40–90 mg/kg ketamine; 5–15 mg/kg xylazine). A mid-longitudinal incision was made on the dorsal surface of cranium (the periosteum was removed). A trephine bur was used to create a circular defect with a diameter of 8 mm at full thickness of 1–1.5 mm. A CPC paste (0.2 g) was placed between two glass plates to yield a thickness of approximately 1 mm. The paste was shaped as a disk with a diameter of 8 mm and placed into the defect. After 4 or 24 weeks, the animals were sacrificed with carbon monoxide . Six groups of scaffolds, with n = 5, required 60 rats.

Histological and micro-CT analyses

For each rat, the implant with surrounding native bone was retrieved. The tissues were fixed for 24 h at 4 °C in 10% phosphate-buffered formalin. Following fixation, implants were analyzed by micro CT (μCT) following previous studies . The rat skull was wrapped in a moistened cotton mesh and placed in a custom-built radiolucent batch scanning acrylic tube. The scans were performed on a Viva40 μCT (Scanco Medical AG), at a voltage of 55 kV, current of 145 μA, and integration time of 314 ms. Voxel size was selected to be isotropic and fixed at 35.5 μm. The scan axis was normal to the subject frontal plane . Subsequently, the whole skull was decalcified in a weak (15%) buffered formic acid and trimmed to a section of 1 mm in front of the defect and 1 mm behind the defect. This strip of skull bone with the defect in the middle was processed and embedded in paraffin. Using a microtome, 5 mm of the samples was cut away to transfer 5 μm thick sections from the central area of the original defect site onto a glass slide. These sections were stained with hematoxylin and eosin (H&E) (Mass Histology Service, Worcester, MA). The new bone and original implantation areas were calculated by Image J (NIH) software. The new bone area fraction was measured as the new bone area/original total implant area.

Statistical analysis

One-way and two-way nonparametric Mann–Whitney tests were performed to detect significant effects of the variables. Tukey’s multiple comparison tests were used at p = 0.05.

Materials and methods

CPC composite scaffold fabrication

TTCP (Ca 4 (PO 4 ) 2 O) was synthesized from a solid-state reaction at 1500 °C between DCPA (CaHPO 4 ) and CaCO 3 (J.T. Baker, Phillipsburg, NJ). The mixture was ground to obtain TTCP particles with sizes of 1–80 μm, with a median particle size of 17 μm. DCPA was ground to obtain particles with sizes of 0.4–3.0 μm, with a median particle size of 1.0 μm. The TTCP and DCPA were then mixed to form the CPC powder. Traditionally, the TTCP/DCPA molar ratio was 1/1 . Recently, a TTCP/DCPA ratio of 1/3 was shown to yield CPC with strength similar to that using 1/1, while the 1/3 ratio had faster dissolution which indicates potentially faster resorption in vivo . Hence, in this study, the CPC control used a TTCP/DCPA ratio of 1/1, while the other groups used a TTCP/DCPA ratio of 1/3. Hence the TTCP/DCPA ratio of the CPC control was the same as the CPC in previous in vivo studies . Previous studies incorporated water-soluble mannitol into CPC to create macropores , and alginate microbeads into CPC as drug and cell carriers . While macroporous CPC was mechanically-weaker than CPC without macropores, chitosan and fibers were incorporated into CPC for reinforcement. Therefore, for mechanical testing, four CPCs were made: (i) CPC control (without mannitol or microbeads); (ii) macroporous CPC with 30% mannitol, 30% alginate microbeads, and 15% chitosan in CPC liquid (referred to as “macroporous CPC”); (iii) same as (ii), but with 15% large-diameter absorbable fibers (“macroporous CPC + large fiber”); (iv) same as (ii), but with 7.5% large-diameter fibers + 7.5% nanofibers (“macroporous CPC + large fiber + nanofiber”).

Alginate hydrogel microbeads were synthesized using a previously-described method . First, the alginate was oxidized to increase its degradability in vivo . Oxidation was performed using sodium periodate at the correct stoichiometric ratio of sodium periodate/alginate to have certain percentages of alginate oxidation . The percentage of oxidation (%) was the number of oxidized uronate residues per 100 uronate units in the alginate chain. Alginate at 7.5% oxidation was synthesized as described previously . Briefly, 1% of sodium alginate was dissolved in water. Then 1.51 mL of 0.25 mol/L sodium periodate (Sigma–Aldrich) was added to 100 mL alginate solution. At 24 h, the oxidization reaction was stopped by adding 1 g of ethylene glycol and then 2.5 g of sodium chloride. The precipitates were re-dissolved and the second precipitates were collected. The oxidized alginate thus obtained was dissolved in saline at a concentration of 1.2%. The solution was loaded into a bead-generating device (Var-J1, Nisco, Zurich, Switzerland). The droplets fell into a well of 100 mmol/L calcium chloride and crosslinked to form microbeads. The microbeads were slightly elongated with a mean length of 335 μm and a mean diameter of 232 μm as measured previously .

A suture fiber (Vicryl, Ethicon, Somerville, NJ) was cut into 3 mm filaments. This suture consisted of fibers braided into a bundle with a diameter of 322 μm, suitable for producing long cylindrical macropores after fiber dissolution in CPC. Chitosan malate (Vanson, Redmond, WA) was mixed with water at a chitosan/(chitosan + water) mass fraction of 15% to form the CPC liquid. Nanofibers of poly( d , l -lactide- co -glycolide) (PLGA) at a poly(lactic acid) (PLA)/poly(glycolic acid) (PGA) ratio of 50/50 were electrospun into non-woven mats, following a previous study . The mat was cut into 3 × 3 mm squares, because a previous study showed that 3 mm fibers in CPC were injectable through a 10-gauge needle . Another study also cut electrospun fibers into 3 mm dimensions for use in CPC . CPC powder and liquid were mixed at a mass ratio of 2:1, and the prescribed amounts of fibers and microbeads were mixed with the CPC–chitosan paste. The mixed composite paste was placed into 3 mm × 4 mm × 25 mm molds and set at 100% humidity for 4 h at 37 °C. The specimens were then demolded and immersed in water at 37 °C for 20 h.

Mechanical testing

A three-point flexural test was performed using a 20 mm span and a displacement rate of 1 mm/min to measure the mechanical properties of CPC. Flexural strength was calculated as S = 3 F max L /(2 bh 2 ), where F max is the maximum load, L is the span, b is specimen width, and h is specimen thickness. Elastic modulus was calculated as E = ( F / c )( L 3 /[4 bh 3 ]), where load F divided by displacement c is the slope of the load–displacement curve in the linear elastic region. Work-of-fracture (toughness) was measured as the area of the load–displacement curve divided by the specimen’s cross-sectional area .

Critical-sized cranial defect model in rats

The mechanical property results showed that the macroporous CPC group (ii) was much weaker than CPC control (i). However, fiber reinforcement rendered the macroporous CPC (iii and iv) to have similar strengths to CPC control. Therefore, for the animal study, the following six groups were tested:

  • (1)

    Control CPC without macropores and microbeads (similar to the CPC in previous in vivo studies, such as Friedman et al. );

  • (2)

    Macroporous CPC + large fiber (30% mannitol, 30% alginate microbeads, 15% chitosan in cement liquid, reinforced with 15% large-diameter Vicryl fibers);

  • (3)

    Macroporous CPC + large fiber + nanofiber (same as 2, except reinforcement with 7.5% Vicryl fibers + 7.5% nanofibers);

  • (4)

    Same as (3), but with rhBMP2 in the CPC matrix;

  • (5)

    Same as (3), but with rhBMP2 in the CPC matrix + rhTGF-β1 in the microbeads;

  • (6)

    Same as (3), but with rhBMP2 in the CPC matrix + VEGF in the microbeads.

rhBMP2, VEGF and rhTGF-β1 were commercially-obtained (PeproTech, Rocky Hill, NJ). Their amounts were 10 μg rhBMP2/defect, 6 μg VEGF/defect, and 200 ng rhTGF-β1/defect, selected following previous studies .

The rat critical-sized cranial defect model was approved by Harvard University (# 24-09) and University of Maryland (# 0909014). NIH procedures were followed. Surgery was performed at Harvard University. Lewis rats (age: 49–55 days, Charles River, Wilmington, MA) were anesthetized by intraperitoneal injection (40–90 mg/kg ketamine; 5–15 mg/kg xylazine). A mid-longitudinal incision was made on the dorsal surface of cranium (the periosteum was removed). A trephine bur was used to create a circular defect with a diameter of 8 mm at full thickness of 1–1.5 mm. A CPC paste (0.2 g) was placed between two glass plates to yield a thickness of approximately 1 mm. The paste was shaped as a disk with a diameter of 8 mm and placed into the defect. After 4 or 24 weeks, the animals were sacrificed with carbon monoxide . Six groups of scaffolds, with n = 5, required 60 rats.

Histological and micro-CT analyses

For each rat, the implant with surrounding native bone was retrieved. The tissues were fixed for 24 h at 4 °C in 10% phosphate-buffered formalin. Following fixation, implants were analyzed by micro CT (μCT) following previous studies . The rat skull was wrapped in a moistened cotton mesh and placed in a custom-built radiolucent batch scanning acrylic tube. The scans were performed on a Viva40 μCT (Scanco Medical AG), at a voltage of 55 kV, current of 145 μA, and integration time of 314 ms. Voxel size was selected to be isotropic and fixed at 35.5 μm. The scan axis was normal to the subject frontal plane . Subsequently, the whole skull was decalcified in a weak (15%) buffered formic acid and trimmed to a section of 1 mm in front of the defect and 1 mm behind the defect. This strip of skull bone with the defect in the middle was processed and embedded in paraffin. Using a microtome, 5 mm of the samples was cut away to transfer 5 μm thick sections from the central area of the original defect site onto a glass slide. These sections were stained with hematoxylin and eosin (H&E) (Mass Histology Service, Worcester, MA). The new bone and original implantation areas were calculated by Image J (NIH) software. The new bone area fraction was measured as the new bone area/original total implant area.

Statistical analysis

One-way and two-way nonparametric Mann–Whitney tests were performed to detect significant effects of the variables. Tukey’s multiple comparison tests were used at p = 0.05.

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Nov 25, 2017 | Posted by in Dental Materials | Comments Off on Bone regeneration via novel macroporous CPC scaffolds in critical-sized cranial defects in rats
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