Jonas Åberg

Calcium phosphates compounds in conjunction with hydrogel as carrier for BMP-2 : A study on ectopic bone formation in rats

Current treatment of fractures often involves the use of bone graft or bone morphogenetic proteins (BMP) to induce fracture healing, especially in patients with a compromised healing capacity. BMP has to be delivered in conjunction with a carrier. Unfortunately, there are drawbacks and limitations with current carriers, including their bovine origin which carries the risk of an immunological response. The physical properties also limit the use to open surgical procedures, as it cannot be injected. New carriers with improved properties are therefore needed. The aim of this study was to assess the ectopic bone forming capability of various calcium phosphate compounds when used in conjunction with a hydrogel as the carrier for BMP-2. Five different ceramic additives were tested, including β-tricalcium phosphate and four types of hydroxyapatite (HAP) (nanoHAP, HAP, clods of HAP >100 μm, and the biomimetic HAP Ostim35®). The compounds were injected into the thigh muscle of rats, where it formed a gel in situ. After 4 weeks bone formation was evaluated by peripheral quantitative computed tomography and histology. The major finding was that the 20 nm nanoHAP yielded a higher bone density than the other additives (P=0.0008, ANOVA with Tukeys multiple comparison test). We hypothesize that the higher bone density induced by nanoHAP might be due to nanocrystals of calcium phosphate acting as direct building blocks for biomineralization.

Premixed acidic calcium phosphate cement: Characterization of strength and microstructure

By using a premixed calcium phosphate cement (CPC), the handling properties of the cement are drastically improved, which is a challenge for traditional injectable CPCs. Previously premixed cements have been based on apatitic cements. In this article, acidic cement has been developed and evaluated. Monocalcium phosphate monohydrate and beta-tricalcium phosphate were mixed with glycerol to form a paste. As the paste does not contain water, no setting reaction starts and thus the working time is indefinite. Powder/liquid ratios (P/L) of 2.25, 3.5 and 4.75 were evaluated. Setting time (ST) and compressive strength (CS) were measured after 1 day, 1 week and 4 weeks in phosphate buffered saline (PBS) solution, and the corresponding microstructure was evaluated using electron microscopy and X-ray diffraction. The ST started when the cements were placed in PBS and ranged from 28 to 75 min, higher P/L gave a lower ST. Higher P/L also gave a higher CS, which ranged from 2 to 16 MPa. The microstructure mainly consisted of monetite, 1-5 microm in grain size. After 4 weeks in PBS, the strength increased. As acidic cements are resorbed faster in vivo, this cement should allow faster bone regeneration than apatitic cements. Premixed cements show a great handling benefit when compared with normal CPCs and can be formulated with similar ST and mechanical properties.

Development of a bioactive implant for repair and potential healing of cranial defects

The repair of complex craniofacial bone defects is challenging and a successful result is dependent on the size of the defect, quality of the soft tissue covering the defect, and choice of reconstruction method. The objective of this study was to develop a bioactive cranial implant that could provide a permanent reconstructive solution to the patient by stimulating bone healing of the defect. In this paper the authors report on the feasibility and clinical results of using such a newly developed device for the repair of a large traumatic and therapy-resistant cranial bone defect. The patient had undergone numerous attempts at repair, in which established methods had been tried without success. A mosaic-designed device was manufactured and implanted, comprising interconnected ceramic tiles with a defined calcium phosphate composition. The clinical outcome 30 months after surgery revealed a restored cranial vault without postoperative complications. Computed tomography demonstrated signs of bone ingrowth. Examination with combined (18)F-fluoride PET and CT provided further evidence of bone healing of the cranial defect.

Influence of water content on hardening and handling of a premixed calcium phosphate cement

Handling of calcium phosphate cements is difficult, where problems often arise during mixing, transferring to syringes, and subsequent injection. Via the use of premixed cements the risk of handling complications is reduced. However, for premixed cements to work in a clinical situation the setting time needs to be improved. The objective of this study is to investigate the influence of the addition of water on the properties of premixed cement. Monetite-forming premixed cements with small amounts of added water (less than 6.8 wt.%) were prepared and the influence on injectability, working time, setting time and mechanical strength was evaluated. The results showed that the addition of small amounts of water had significant influence on the properties of the premixed cement. With the addition of just 1.7 wt.% water, the force needed to extrude the cement from a syringe was reduced from 107 (±15) N to 39 (±9) N, the compression strength was almost doubled, and the setting time decreased from 29 (±4) min to 19 (±2) min, while the working time remained 5 to 6h. This study demonstrates the importance of controlling the water content in premixed cement pastes and how water can be used to improve the properties of premixed cements.

In Vivo Evaluation of an Injectable Premixed Radiopaque Calcium Phosphate Cement

In this work a radiopaque premixed calcium phosphate cement (pCPC) has been developed and evaluated in vivo. Radiopacity was obtained by adding 0–40 % zirconia to the cement paste. The effects of zirconia on setting time, strength and radiopacity were evaluated. In the in vivo study a 2 by 3.5 mm cylindrical defect in a rat vertebrae was filled with either the pCPC, PMMA or bone chips. Nano-SPECT CT analysis was used to monitor osteoblast activity during bone regeneration. The study showed that by adding zirconia to the cement the setting time becomes longer and the compressive strength is reduced. All materials evaluated in the in vivo study filled the bone defect and there was a strong osteoblast activity at the injury site. In spite of the osteoblast activity, PMMA blocked bone healing and the bone chips group showed minimal new bone formation. At 12 weeks the pCPC was partially resorbed and replaced by new bone with good bone ingrowth. The radiopaque pCPC may be considered to be used for minimal invasive treatment of vertebral fractures since it has good handling, radiopacity and allows healing of cancellous bone in parallel with the resorption of the cement.

Influence of particle size on hardening and handling of a premixed calcium phosphate cement

Premixed calcium phosphate cements (pCPC) have been developed to circumvent problems related to mixing and transfer of cements in the operating room. In addition, by using pCPC the short working times generally associated with conventional water-mixed cements are avoided. In this work, the influence of particle size on handling and hardening characteristics of a premixed monetite cement has been assessed. The cements were evaluated with respect to their injectability, setting time and compressive strength. It was found that cements with smaller particle sizes were more difficult to inject and had higher compressive strength. Regarding setting time, no clear trend could be discerned. The addition of granules made the cements easier to inject, but setting time was prolonged and lower strengths were obtained. The main findings of this work demonstrate that particle size can be used to control handling and physical properties of premixed cements and that previous knowledge from water-based CPC, regarding effects of particle size, is not directly applicable to premixed CPC.

FGF1 containing biodegradable device with peripheral nerve grafts induces corticospinal tract regeneration and motor evoked potentials after spinal cord resection

PURPOSE Repairing the spinal cord with peripheral nerve grafts (PNG) and adjuvant acidic fibroblast growth factor (FGF1) has previously resulted in partial functional recovery. To aid microsurgical placement of PNGs, a graft holder device was previously developed by our group. In hope for a translational development we now investigate a new biodegradable graft holder device containing PNGs with or without FGF1. METHODS Rats were subjected to a T11 spinal cord resection with subsequent repair using twelve white-to-grey matter oriented PNGs prepositioned in a biodegradable device with or without slow release of FGF1. Animals were evaluated with BBB-score, electrophysiology and immunohistochemistry including anterograde BDA tracing. RESULTS Motor evoked potentials (MEP) in the lower limb reappeared at 20 weeks after grafting. MEP responses were further improved in the group treated with adjuvant FGF1. Reappearance of MEPs was paralleled by NF-positive fibers and anterogradely traced corticospinal fibers distal to the injury. BBB-scores improved in repaired animals. CONCLUSIONS The results continue to support that the combination of PNGs and FGF1 may be a regeneration strategy to reinnervate the caudal spinal cord. The new device induced robust MEPs augmented by FGF1, and may be considered for translational research.

Biocompatibility and resorption of a radiopaque premixed calcium phosphate cement.

Calcium phosphate cements (CPC) are used as bone void filler in various orthopedic indications; however, there are some major drawbacks regarding mixing, transfer, and injection of traditional CPC. By using glycerol as mixing liquid, a premixed calcium phosphate cement (pCPC), some of these difficulties can be overcome. In the treatment of vertebral fractures the handling characteristics need to be excellent including a high radio-opacity for optimal control during injection. The aim of this study is to evaluate a radiopaque pCPC regarding its resorption behavior and biocompatibility in vivo. pCPC and a water-based CPC were injected into a Ø 4-mm drilled femur defect in rabbits. The rabbits were sacrificed after 2 and 12 weeks. Cross sections of the defects were evaluated using histology, electron microscopy, and immunohistochemical analysis. Signs of inflammation were evaluated both locally and systemically. The results showed a higher bone formation in the pCPC compared to the water-based CPC after 2 weeks by expression of RUNX-2. After 12 weeks most of the cement had been resorbed in both groups. Both materials were considered to have a high biocompatibility since no marked immunological response was induced and extensive bone ingrowth was observed. The conclusion from the study was that pCPC with ZrO(2) radiopacifier is a promising alternative regarding bone replacement material and may be suggested for treatment of, for example, vertebral fractures based on its high biocompatibility, fast bone ingrowth, and good handling properties.

Calcium Sulfate Spinal Cord Scaffold: A Study on Degradation and Fibroblast Growth Factor 1 Loading and Release

Currently, there is no regenerative strategy for the spinal cord that is part of clinical standard of core. Current paths usually include combinations of scaffold materials and active molecules. In a recent study, a permanent dental resin scaffold for treatment of spinal cord injury was designed. The results from studies on rats were promising. However, for potential clinical use, a biodegradable scaffold material that facilitates drug delivery and the regeneration of the spinal cord needs to be developed. Also a biodegradable material is expected to allow a better evaluation of the efficacy of the surgical method. In this article, the suitability of hardened calcium sulfate cement (CSC) for use as degradable spinal cord scaffolds is investigated in bench studies and in vitro studies. Compressive strength, degradation and microstructure, and the loading capability of heparin-activated fibroblast growth factor 1 (FGF1) via soaking were evaluated. The CSC could easily be injected into the scaffold mold and the obtained scaffolds had sufficient strength to endure the loads applied during surgery. When hardened, the CSC formed a porous microstructure suitable for loading of active substances. It was shown that 10 min of FGF1 soaking was enough to obtain a sustained active FGF1 release for 20–35 days. The results showed that CSC is a promising material for spinal cord scaffold fabrication, since it is biodegradable, has sufficient strength, and allows loading and controlled release of active FGF1.

Setting mechanisms of an acidic premixed calcium phosphate cement

Premixed calcium phosphate cements (pCPC), where glycerol is used instead of water as mixing liquid, present better handling characteristics than water-based cements. However, the setting mechanisms of pCPC have not been described thoroughly. The aim of this paper is to increase the understanding of the setting mechanism of pCPC. The investigated cement starts to set when glycerol is exchanged with water via diffusion of glycerol out to the surrounding body fluid and water into the material. To better understand the water-glycerol exchange a method was developed where the setting depth of the cement was measured over time. Thermo gravimetric analysis (TGA) was used to determine the liquid exchange rate during setting. To study the influence of temperature on the crystalline end product, pCPC and water-mixed calcium phosphate cement (wCPC) were set at different temperatures and analyzed with X-ray diffraction (XRD). The setting depth measurements showed that the set layer of the pCPC grew with a speed proportional to t0.51 at 37°C. TGA results furthermore showed that less than 10% of the glycerol remained after 16 hours. Setting of pCPC at different temperatures showed that mainly brushite was formed at 5°C, a mixture of brushite and monetite at 21°C and mainly monetite at 37°C. It furthermore showed that brushite was the main phase after setting of wCPC, but some monetite was present in these cements. The study presents a new method for evaluation of pCPC that increases the understanding of their setting mechanism. Furthermore, the XRD results indicate that storage at 5°C could improve the shelf life of acidic pCPC.