Publications

At Moroxite F, we bring science and innovation together. Our research group, TERM at LU has 50+ published scientific articles related to the work in Moroxite F. Please find selected publications below.

Hydroxyapatite (HA) has been widely used as a bone substitute and more recently as a carrier for local delivery of bone targeted drugs. Majority of the approved HA based biomaterials and drug carriers comprise of micrometer sized particulate HA (mHA) or granules and can therefore only be used for extracellular drug release. This shortcoming could be overcome with the use of cell penetrating HA nanoparticles (nHA) but a major concern with the clinical use of nHA is the lack of data on its in vivo biodistribution after implantation. In this study, we aimed to study the in vivo biodistribution of locally implanted nHA in a clinically relevant tibial void in rats and compare it with mHA or a combination of mHA and nHA. To enable in vivo tracking, HA particles were first labelled with 14C-zoledronic acid (14C-ZA), known to have a high binding affinity to HA. The labelled particles were then implanted in the animals and the radioactivity in the proximal tibia and vital organs was detected at various time points (Day 1, 7 and 28) post-implantation using scintillation counting. The local distribution of the particles in the bone was studied with micro-CT. We found that majority (>99.9%) of the implanted HA particles, irrespective of the size, stayed locally at the implantation site even after 28 days and the findings were confirmed using micro-CT. Less than 0.1% radioactivity was observed in the kidney and the spleen at later time points of day 7 and 28. No pathological changes in any of the vital organs could be observed histologically. This is the first longitudinal in vivo HA biodistribution study showing that the local implantation of nHA particles in bone is safe and that nHA could potentially be used for localized drug delivery.

Cement augmentation of pedicle screws is one of the most promising approaches to enhance the anchoring of screws in the osteoporotic spine. To date, there is no ideal cement for pedicle screw augmentation. The purpose of this study was to investigate whether an injectable, bioactive, and degradable calcium sulfate/hydroxyapatite (CaS/HA) cement could increase the maximum pull-out force of pedicle screws in osteoporotic vertebrae. Herein, 17 osteoporotic thoracic and lumbar vertebrae were obtained from a single fresh-frozen human cadaver and instrumented with fenestrated pedicle screws. The right screw in each vertebra was augmented with CaS/HA cement and the un-augmented left side served as a paired control. The cement distribution, interdigitation ability, and cement leakage were evaluated using radiographs. Furthermore, pull-out testing was used to evaluate the immediate mechanical effect of CaS/HA augmentation on the pedicle screws. The CaS/HA cement presented good distribution and interdigitation ability without leakage into the spinal canal. Augmentation significantly enhanced the maximum pull-out force of the pedicle screw in which the augmented side was 39.0% higher than the pedicle-screw-alone side. Therefore, the novel biodegradable biphasic CaS/HA cement could be a promising material for pedicle screw augmentation in the osteoporotic spine.

Background

The treatments for trochanteric fractures try to regain early mobility and limit morbidity and risk of re-operations. The most currently used dynamic hip screw (DHS) and the proximal femoral nail (PFN) are both with pros and cons. We aimed to assess the comparative effectiveness of these interventions for trochanteric fractures by evaluating the surgical performance and postoperative outcomes.

Methods

PubMed, Web of Science and Cochrane Central Register were searched for RCTs comparing DHS and PFN for trochanteric fractures. All selected studies and the risk of bias were assessed. Clinical data including operative time, intra-operative blood loss, intra-operative fluoroscopy time, successful closed reduction and complications like nonunion, implant failure and re-operation were recorded. Random-effects models were used in Review Manager software, and GRADE was applied for the interpretation of the evidence.

Results

From 286 identified trials, twelve RCTs including 1889 patients were eligible for inclusion; six RCTs directly comparing DHS with PFN, while other six compared DHS with proximal femoral nail antirotation (PFNA). Compared to DHS, PFN had shorter operative time and led to less intra-operative blood loss. However, DHS need less intra-operative fluoroscopy time than PFN. No difference was seen for the achievement of closed reduction. For risk of postoperative complications, no difference was seen between PFN and DHS for non-union, risk of implant failure and revision surgery.

Conclusions

PFN(A) resulted in a shorter operative time and less intra-operative blood loss compared to DHS. However, no difference was seen for postoperative complications.

A New Augmentation Method for Improved Screw Fixation in Fragile Bone

MARCH 2022 | FRONTIERS IN BIOENGINEERING AND BIOTECHNOLOGY

Pertrochanteric fractures (TF) due to osteoporosis constitute nearly half of all proximal femur fractures. TFs are treated with a surgical approach and fracture fixation is achieved using metallic fixation devices. Poor quality cancellous bone in osteoporotic patients makes anchorage of a fixation device challenging, which can lead to failure of the fracture fixation. Methods to reinforce the bone-implant interface using bone cement (PMMA) and other calcium phosphate cements in TFs have been described earlier but a clear evidence on the advantage of using such biomaterials for augmentation is weak. Furthermore, there is no standardized technique for delivering these biomaterials at the bone-implant interface. In this study, we firstly describe a method to deliver a calcium sulphate/hydroxyapatite (CaS/HA) based biomaterial for the augmentation of a lag-screw commonly used for TF fixation. We then used an osteoporotic Sawbones model to study the consequence of CaS/HA augmentation on the immediate mechanical anchorage of the lag-screw to osteoporotic bone. Finally, as a proof-of-concept, the method of delivering the CaS/HA biomaterial at the bone-implant interface as well as spreading of the CaS/HA material at this interface was tested in patients undergoing treatment for TF as well as in donated femoral heads. The mechanical testing results indicated that the CaS/HA based biomaterial increased the peak extraction force of the lag-screw by 4 times compared with un-augmented lag-screws and the results were at par with PMMA. The X-ray images from the patient series showed that it was possible to inject the CaS/HA material at the bone-implant interface without applying additional pressure and the CaS/HA material spreading was observed at the interface of the lag-screw threads and the bone. Finally, the spreading of the CaS/HA material was also verified on donated femoral heads and micro-CT imaging indicated that the entire length of the lag-screw threads was covered with the CaS/HA biomaterial. In conclusion, we present a novel method for augmenting a lag-screw in TFs, which could potentially reduce the risk of fracture fixation failure and reoperation in fragile osteoporotic patients

This research aims to investigate nonionic hyperbranched polyesters (HBPs) derived from indole and lignin resources as new nontoxic antimicrobial coatings. Three nonionic HBPs with zero to two methoxy ether substituents on each benzene ring in the polymer backbones were synthesized by melt-polycondensation of three corresponding AB2 monomers. The molecular structures and thermal properties of the obtained HBPs were characterized by gel permeation chromatography, nuclear magnetic resonance spectroscopy, Fourier transform infrared spectroscopy, thermogravimetric analysis, and differential scanning calorimetry analyses. These HBPs were conveniently spin-coated on a silicon substrate, which exhibited significant antibacterial effect against Gram-negative (Escherichia coli and Pseudomonas aeruginosa) and Gram-positive bacteria (Staphylococcus aureus and Enterococcus faecalis). The presence of methoxy substituents enhanced the antimicrobial effect, and the resulting polymers showed negligible leakage in water. Finally, the polymers with the methoxy functionality exhibited excellent biocompatibility according to the results of hemolysis and MTT assay, which may facilitate their biomedical applications.

Hip Fractures: A Biomechanical Analysis Of Fracture Strength Prediction, Prevention, And Repair

OCTOBER 2021 | DOCTORAL THESIS

Due to the aging population, hip fracture incidence has been increasing over the past decades. Measurements of bone mineral density with dual energy X-ray absorptiometry are the gold standard for hip fracture risk assessment, where patients with a low bone density have a high risk of fracture. However, many people that are not diagnosed to be at risk, still fracture their hip. Calculations of bone strength using subject-specific finite element (FE) models, can improve fracture risk prediction, but further improvement is required.
Patients with a high fracture risk are often prescribed pharmaceutical treatment in order to increase bone density systemically. As systemic response to treatment is limited, other options to prevent fractures by improving the bone strength are investigated. One of those options is the injection of biomaterials in the femoral neck. In case of a hip fracture due to a low-energy fall, total hip replacement is generally preferred over joint-preserving methods like fixation using a dynamic hip screw. Screw fixation comes with a risk of screw instability, especially in low-density bone. Bone cements can be used to improve fixation of orthopaedic implants and fracture fixation devices. Calcium sulphate/hydroxyapatite (CaS/HA) is an injectable biomaterial that has been used, for example, to reinforce collapsed vertebrae and to stabilize wrist fractures.
The work presented in the thesis aims to improve fracture risk prediction, and fracture prevention and repair methods with use of CaS/HA. This is achieved through a combination of experimental mechanical tests at organ and tissue scale, and development and thorough validation of FE models of the proximal femur.
In the first part of this thesis, 12 cadaveric femora were used in an experiment where the bones were loaded until fracture in a configuration developed to replicate a fall to the side. During loading, high-speed cameras were used to image both the medial and lateral side of the femoral neck allowing for full-field strain measurements using digital image correlation. The femora were imaged with clinical CT before and micro-CT before and after mechanical testing. Using the acquired CT images, FE models were developed at two different resolutions to determine their ability to capture the fracture force, fracture location and surface strains. The FE models based on the clinical CT images were able to accurately capture the fracture force and identify regions where the bone would fracture. These models could also capture the strains with high accuracy. However, the strains were not predicted as accurately in regions with high surface irregularity. The models based on the micro-CT images could show with higher accuracy how the strains were distributed around local porosity (e.g., due to vascularization) in the femoral neck and how these influenced the fracture pattern.
The thesis continues with an investigation of fracture prevention and repair methods through the use of CaS/HA. The ability of CaS/HA to increase the fracture strength of the proximal femur for fracture prevention and its ability to stabilize a dynamic hip screw used for fracture repair was investigated. The increase in fracture strength was investigated using FE models. These models showed that CaS/HA can increase the fracture strength of the femur approximately 20% when injected close to the cortex in the lateral neck. Pullout tests using a dynamic hip screw were performed on synthetic bone blocks and femoral heads from hip fracture patients. In the synthetic blocks, CaS/HA significantly increased the pullout strength. However, in the human bone the stability of the screw was not improved, because the cement could not easily spread into the threads of the screws. The mechanical behaviour of CaS/HA and bone was further investigated using high-resolution synchrotron X-ray tomography. Cylindrical trabecular bone specimens with and without CaS/HA were imaged with tomography during in-situ loading of the samples. The images revealed that CaS/HA reinforced the bone, and that CaS/HA is a brittle material that will crack before the bone.
To conclude, in this thesis FE models are presented showing accurate prediction of fracture strength, which can be used for improved fracture risk assessments. Furthermore, the work provides insight in how CaS/HA behaves mechanically and how it can be used to increase the fracture strength and to stabilize fixation devices in the femur, improving fracture prevention and fracture repair methods.

The bone tissue formed at the contact interface with metallic implants, particularly its 3D microstructure, plays a pivotal role for the structural integrity of implant fixation. X-ray tomography is the classical imaging technique used for accessing microstructural information from bone tissue. However, neutron tomography has shown promise for visualising the immediate bone-metal implant interface, something which is highly challenging with X-rays due to large differences in attenuation between metal and biological tissue causing image artefacts. To highlight and explore the complementary nature of neutron and X-ray tomography, proximal rat tibiae with titanium-based implants were imaged with both modalities. The two techniques were compared in terms of visualisation of different material phases and by comparing the properties of the individual images, such as the contrast-to-noise ratio. After superimposing the images using a dedicated image registration algorithm, the complementarity was further investigated via analysis of the dual modality histogram, joining the neutron and X-ray data. From these joint histograms, peaks with well-defined grey value intervals corresponding to the different material phases observed in the specimens were identified and compared. The results highlight differences in how neutrons and X-rays interact with biological tissues and metallic implants, as well as the benefits of combining both modalities. Future refinement of the joint histogram analysis could improve the segmentation of structures and tissues, and yield novel information about specimen-specific properties such as moisture content.

Augmenting A Dynamic Hip Screw With A Calcium Sulfate/Hydroxyapatite Biomaterial

MAY 2021 | MEDICAL ENGINEERING AND PHYSICS

Internal fixation failure in hip fractures can lead to reoperation. Calcium sulfate/hydroxyapatite (CaS/HA) is a biomaterial that can be used for augmenting fracture fixation. We aimed to determine whether an injection of 2 ml CaS/HA increases the fixation of a dynamic hip screw inserted in synthetic and human trabecular bone. The study consists of two parts: 1) synthetic bone blocks (n=74), with three subgroups: empty (cannulated screw, no injection), cannulated, and fenestrated; and 2) osteoporotic human femoral heads (n=29), with the same subgroups. The heads were imaged using µCT. Bone volume fraction, insertion angle, and head diameter were measured. Pullout tests were performed and peak force, stiffness, and work were measured. The fenestrated group showed increases in pullout strength compared to no injection in the synthetic blocks. The cannulated group showed a higher pullout strength in low-density blocks. In the femoral heads, the variation was larger and there were no significant differences between groups. The bone volume fraction correlated with the peak force and work, and the insertion angle correlated with the stiffness. CaS/HA can improve the fixation of a dynamic hip screw. For clinical use, spreading of the material around the threads of the screw must be ensured

Ceramic biomaterials are promising alternatives to bone autografts. However, limited bioactivity affects their performance. Therefore, bioactive molecules and cells are often added to enhance their performance. Exosomes have emerged as cell-secreted vesicles, delivering proteins, lipids, and nucleic acids in a paracrine/endocrine fashion. We studied two complementary aspects required for exosome activity/therapy using purified exosomes: first, the intracellular uptake of labeled exosomes and second, the influence of delivered exosomes on cell behavior. Origin-specific differences in the characteristics of purified exosomes, quantification of time-dependent intracellular uptake of PKH-26-labeled exosomes by mesenchymal stem cells (MSCs) and preosteoblasts, and influence on cell behavior were evaluated. Furthermore, exosomes from osteoblasts and MSCs cultured under normal and osteogenic environments were isolated. There is little data available on the concentration and dose of exosomes required for bone regeneration. Therefore, equal amounts of quantified exosomes were implanted in vivo in rat tibia critical defects using a calcium sulfate–nano-hydroxyapatite nanocement (NC) bone filler as the carrier. Bone regeneration was quantified using micro-computed tomography and histology. Along with inducing early maturation and mineral deposition by primary preosteoblasts in vitro, exosome treatment also demonstrated a positive effect on bone mineralization in vivo. Our study concludes that providing a local delivery of exosomes loaded onto a slowly resorbing NC bone filler can provide a potential alternate to autografts as a bone substitute.

Bone morphogenic proteins (BMPs) are the only true osteoinductive molecules. Despite being tremendously potent, their clinical use has been limited for reasons including supraphysiological doses, suboptimal delivery systems, and the pro-osteoclast effect of BMPs. Efforts to achieve spatially controlled bone formation using BMPs are being made. We demonstrate that a carrier consisting of a powder of calcium sulfate/hydroxyapatite (CaS/HA) mixed with bone active molecules provides an efficient drug delivery platform for critical femoral defect healing in rats. The bone-active molecules were composed of osteoinductive rhBMP-2 and the bisphosphonate, and zoledronic acid (ZA) was chosen to overcome BMP-2–induced bone resorption. It was demonstrated that delivery of rhBMP-2 was necessary for critical defect healing and restoration of mechanical properties, but codelivery of BMP-2 and ZA led to denser and stronger fracture calluses. Together, the CaS/HA biomaterial with rhBMP-2 and/or ZA can potentially be used as an off-the-shelf alternative to autograft bone.

Bone is a dynamic tissue with a quarter of the trabecular and a fifth of the cortical bone being replaced continuously each year in a complex process that continues throughout an individual’s lifetime. Bone has an important role in homeostasis of minerals with non-stoichiometric hydroxyapatite bone mineral forming the inorganic phase of bone. Due to its crystal structure and chemistry, hydroxyapatite (HA) and related apatites have a remarkable ability to bind molecules. This review article describes the accretion of trace elements in bone mineral giving a historical perspective. Implanted HA particles of synthetic origin have proved to be an efficient recruiting moiety for systemically circulating drugs which can locally biomodulate the material and lead to a therapeutic effect. Bone mineral and apatite however also act as a waste dump for trace elements and drugs, which significantly affects the environment and human health.

Osteoporosis often leads to fragility fractures of the hip, resulting in impaired quality of life and increased mortality. Augmenting the proximal femur could be an attractive option for prevention of fracture or fixation device failure. We describe a tissue engineering based strategy to enhance long-term bone formation in the femoral neck of osteoporotic rats by locally delivering bioactive molecules; recombinant human bone morphogenic protein-2 (rhBMP-2), and zoledronic acid (ZA) by using a calcium sulfate/ hydroxyapatite (CaS/HA) biomaterial. A defect was created by reaming the femoral neck canal of osteoporotic (OVX) rats and they were treated as follows: G1. Empty, G2. CaS/HA, G3. CaS/HA+Systemic ZA, G4. CaS/HA+Local ZA, and G5. CaS/HA+Local ZA+rhBMP-2. Bone formation was evaluated 6 months after treatment. Further, radioactively labeled 14C-ZA was used to study the bioavailability of ZA at the defect location, which was determined by using scintillation counting. Micro-CT indicated significantly higher bone volume in groups G4 and G5 compared with the other treatment groups. This was confirmed qualitatively by histological assessment. Addition of rhBMP-2 gave no additional benefit in this model. Local delivery of ZA performed better than systemic administration of ZA. Mechanical testing showed no differences between the groups, likely reflecting that the addition of bioactive molecules had limited effect on cortical bone or the choice of mechanical testing setup was not optimal. Scintillation counting revealed higher amounts of 14C-ZA present in the treated leg of G4 compared with its contralateral control and compared with G3, indicating that local ZA delivery can be used to achieve high local concentrations without causing a systemic effect. This long-term study shows that local delivery of ZA using a CaS/HA carrier can regenerate cancellous bone in the femoral neck canal and has clear implications for enhancing implant integration and fixation in fragile bone.

Publication List

 

    1. Liu Y., et al., Longitudinal in vivo biodistribution of nano and micro sized hydroxyapatite particles implanted in a bone defect. Fronters in Bioengineering and Biotechnology. December 2022 https://doi.org/10.3389/fbioe.2022.1076320
    2. Xinggui Tian., et al., Evaluation of an Injectable Biphasic Calcium Sulfate/Hydroxyapatite Cement for the Augmentation of Fenestrated Pedicle Screws in Osteoporotic Vertebrae: A Biomechanical Cadaver Study. Journal of Functional Biomaterials, December 2022. https://doi.org/10.3390/jfb13040269
    3. Xu, H., Liu, Y., Sezgin, E.A. et al.Comparative effectiveness research on proximal femoral nail versus dynamic hip screw in patients with trochanteric fractures: a systematic review and meta-analysis of randomized trials. Journal of Orthopaedic Surgery and Research. https://doi.org/10.1186/s13018-022-03189-z 
    4. Raina D B., et al., A New Augmentation Method for Improved Screw Fixation in Fragile Bone. Frontiers in Bioengineering and Biotechnology, 2022 https://doi.org/10.3389/fbioe.2022.816250
    5. Erdem Aras Sezgin., et al., A combined fracture and mortality risk index useful for treatment stratification in hip fragility fractures. Joint Diseases and Related Surgery, 2021 https://jointdrs.org/full-text/1299/eng
    6. Elin Törnquist., et al., Dual modality neutron and x-ray tomography for enhanced image analysis of the bone-metal interface. Physics in Medicine & Biology 2021  https://doi.org/10.1088/1361-6560/ac02d4
    7. Joeri Kok., et al., Augmenting a dynamic hip screw with a calcium sulfate/hydroxyapatite biomaterial. Medical Engineering & Physics, 2021 https://doi.org/10.1016/j.medengphy.2021.05.006
    8. Arun K Teotia., et al., Exosome-Functionalized Ceramic Bone Substitute Promotes Critical-Sized Bone Defect Repair in Rats. ACS Publications https://doi.org/10.1021/acsabm.1c00311
    9. Raina D B, Matuszewski L M, Vater C, Bolte J, Isaksson H, Lidgren L, Tägil M, Zwingenberger S. A Facile One-Stage Treatment of Critical Bone Defects Using a Calcium Sulphate/Hydroxyapatite Biomaterial Providing Spatio-Temporal Delivery of Bone Morphogenic Protein-2 and Zoledronic Acid. 2020 https://doi.org/10.1126/sciadv.abc1779
    10. Raina, D.B., et al., Long-Term Response to a Bioactive Biphasic Biomaterial in the Femoral Neck of Osteoporotic Rats. Tissue Engineering Part A, 2020. 26(19-20): p. 1042-1051.
    11. Raina, D.B., et al., Bone mineral as a drug-seeking moiety and a waste dump. Bone & Joint Research, 2020. 9(10): p. 709-718.
    12. Raina, D.B., et al., Synthetic hydroxyapatite: a recruiting platform for biologically active molecules. Acta Orthopaedica, 2020. 91(2): p. 126-132.
    13. Raina, D.B., et al., Synthesis and Characterization of a Biocomposite Bone Bandage for Controlled Delivery of Bone-Active Drugs in Fracture Non-unions. ACS Biomaterials Science & Engineering, 2020. 6(5): p. 2867-2878.
    14. Mathavan, N., et al., Longitudinal in vivo monitoring of callus remodeling in BMP-7- and Zoledronate-treated fractures.Journal of Orthopaedic Research, 2020. 38(9): p. 1905-1913.
    15. Teotia, A.K., et al., Composite bilayered scaffolds with bio-functionalized ceramics for cranial bone defects: An in vivo evaluation.Multifunctional Materials, 2019. 2(1): p. 014002.
    16. Raina, D.B., et al., Guided tissue engineering for healing of cancellous and cortical bone using a combination of biomaterial based scaffolding and local bone active molecule delivery. Biomaterials, 2019. 188: p. 38-49.
    17. Raina, D.B., et al., Biomodulation of an implant for enhanced bone-implant anchorage. Acta Biomaterialia, 2019. 96: p. 619-630.
    18. Mathavan, N., et al., 18F-fluoride as a prognostic indicator of bone regeneration. Acta Biomaterialia, 2019. 90: p. 403-411.
    19. Kok, J., et al., Fracture strength of the proximal femur injected with a calcium sulfate/hydroxyapatite bone substitute. Clinical Biomechanics, 2019. 63: p. 172-178.