Selected Publications

Abstract. Introduction: biomaterials combined with antibiotics are routinely used for the management of bone infections. After eluting high concentrations of antibiotics during the first week, sub-inhibitory concentrations of antibiotics may lead to late repopulation of recalcitrant bacteria. Recent studies have shown that systemically given antibiotics like tetracycline and rifampicin (RIF) could seek and bind to locally implanted hydroxyapatite (HA). The aim of this in vivo study was to test if systemically administered rifampicin could replenish HA-based biomaterials with or without prior antibiotic loading to protect the material from late bacterial repopulation. Methods: in vivo accretion of systemically administered RIF to three different types of HA-based materials was tested. In group 1, nano (n)- and micro (m)-sized HA particles were used, while group 2 consisted of a calcium sulfate/hydroxyapatite (CaS/HA) biomaterial without preloaded antibiotics gentamycin (GEN) or vancomycin (VAN), and in group 3, the CaS/HA material contained GEN (CaS/HA+GEN) or VAN (CaS/HA+VAN). The above materials were implanted in an abdominal muscle pouch model in rats, and at 7 d post-surgery, the animals were assigned to a control group (i.e., no systemic antibiotic) and a test group (i.e., animals receiving one single intraperitoneal injection of RIF each day (4 mg per rat) for 3 consecutive days). Twenty-four hours after the third injection, the animals were sacrificed and the implanted pellets were retrieved and tested against Staphylococcus aureus ATCC 25923 in an agar diffusion assay. After overnight incubation, the zone of inhibition (ZOI) around the pellets were measured. Results: in the control group, 2/6 CaS/HA+GEN pellets had a ZOI, while all other harvested pellets had no ZOI. No pellets from animals in test group 1 had a ZOI. In test group 2, 10/10 CaS/HA pellets showed a ZOI. In test group 3, 5/6 CaS/HA+GEN and 4/6 CaS/HA+VAN pellets showed a ZOI. Conclusions: in this proof-of-concept study, we have shown that a locally implanted biphasic CaS/HA carrier after 1 week can be loaded by systemic RIF administration and exert an antibacterial effect. Further in vivo infection models are necessary to validate our findings.

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.

Osteosarcoma is a malignant cancer of the bone mainly affecting adolescents. Despite progress, the clinical management of osteosarcoma is still challenging. With the current chemotherapy protocol being used for more than 30 years, the number of poor responders is increasing. Although new treatments have been explored since then, no improved tumor eradication effect have been found. In the present thesis, we have developed a new treatment method for osteosarcoma, using hydroxyapatite (HA) based materials as a platform for local delivery of cytostatics. Doxorubicin (DOX), a cornerstone osteosarcoma drug, was chosen as a drug candidate, due to its binding capacity to HA. Different types of HA-based biomaterials were tested for local or targeted delivery of DOX. The efficacy of the developed system was evaluated in-vitro, in osteosarcoma cells as well as in-vivo, in mice bearing an aggressive osteosarcoma.
In Study 1, a clinically approved calcium sulphate (CaS)/HA biomaterial achieved a sustained and controlled release of DOX up to 28 days, both in-vitro and in-vivo. Compared to no treatment or the clinical standard with systemic DOX administration, the local delivery of DOX using a CaS/HA biomaterial significantly hindered tumor progression by inhibiting angiogenesis and cell proliferation.
In Study 2, we investigated the physicochemical interactions between DOX and different sizes of HA particles, both in-vitro and in-vivo. When delivered by HA nanoparticles, DOX is routed to the mitochondria causing insufficient ATP synthesis, less cell migration and cell apoptosis. This leads to stronger in-vivo tumor eradication compared to systemic administration of DOX. Furthermore, nHA mediated delivery of DOX may prevent further metastases in- vivo, which was indirectly verified by PET/CT data.
In Study 3, HA particles (nHA, mHA or n/mHA) were labelled with carbon 14 (14C) to detect particle migration in- vivo. During the observational time of 28 days, the majority (>99.9%) of implanted HA particles, irrespective of the size, stayed in the implantation site (proximal tibia), without migrating to other vital organs. No pathological changes were detected in the vital organs.
In summary, we describe a new and efficient method to supplement osteosarcoma treatment, with a possible rapid translational potential, using clinically approved constituents. By using a hydroxyapatite-based biomaterial, DOX could be routed to the tumor site, more efficiently and with less side effects compared to systemic administration. The chemical interaction between DOX and HA lead to a sustained and controlled DOX release which further improved its tumor eradication effect. When using HA nanoparticles, DOX could be directed to the mitochondria causing tumor cell starvation, reduced migration and apoptosis, jointly leading to improved tumor eradication. The local administration of HA particles, irrespective of size, was confirmed as safe without damage to vital organs. In the future, chemotherapeutics with multi-release profile potentially could be applied by using a combination of nHA and mHA.

Aim

There is a lack of biomaterial-based carriers for the local delivery of rifampicin (RIF), one of the cornerstone second defence antibiotics for bone infections. RIF is also known for causing rapid development of antibiotic resistance when given as monotherapy. This in vitro study evaluated a clinically used biphasic calcium sulphate/hydroxyapatite (CaS/HA) biomaterial as a carrier for dual delivery of RIF with vancomycin (VAN) or gentamicin (GEN).

Methods

The CaS/HA composites containing RIF/GEN/VAN, either alone or in combination, were first prepared and their injectability, setting time, and antibiotic elution profiles were assessed. Using a continuous disk diffusion assay, the antibacterial behaviour of the material was tested on both planktonic and biofilm-embedded forms of standard and clinical strains of Staphylococcus aureus for 28 days. Development of bacterial resistance to RIF was determined by exposing the biofilm-embedded bacteria continuously to released fractions of antibiotics from CaS/HA-antibiotic composites.

Results

Following the addition of RIF to CaS/HA-VAN/GEN, adequate injectability and setting of the CaS/HA composites were noted. Sustained release of RIF above the minimum inhibitory concentrations of S. aureus was observed until study endpoint (day 35). Only combinations of CaS/HA-VAN/GEN + RIF exhibited antibacterial and antibiofilm effects yielding no viable bacteria at study endpoint. The S. aureus strains developed resistance to RIF when biofilms were subjected to CaS/HA-RIF alone but not with CaS/HA-VAN/GEN + RIF.

Conclusion

Our in vitro results indicate that biphasic CaS/HA loaded with VAN or GEN could be used as a carrier for RIF for local delivery in clinically demanding bone infections.

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.

Calcium sulfate/hydroxyapatite (CaS/HA) biomaterials have been investigated for use in several orthopedic applications. However, the mechanical interactions between the composite of CaS/HA and bone at the microscale are still unknown. The aim of this study was to determine if and how augmentation with CaS/HA alters the fracture behavior of bone. Eleven cylinders of trabecular bone were drilled from human femoral heads and cleaned from bone marrow. Among them, five cylinders were injected with CaS/HA to generate composite specimens, while the others were kept intact. One extra specimen of pure CaS/HA was prepared. All specimens were compressed in situ using synchrotron X-ray tomography and imaged at ∼2% strain intervals. Structural properties were calculated from the images in unloaded state and mechanical properties were determined from the load-curves. CaS/HA alone displayed the highest peak force and stiffness and the lowest strain at fracture. All composite specimens had a higher peak force than the pure bone specimens and the composite specimens had higher toughness than the pure CaS/HA specimen. Furthermore, the fracture behavior was analyzed further to characterize the local deformations. The pure bone specimens presented damage in multiple trabeculae and the CaS/HA specimen displayed sharp transition in strains, with low strain in one load step and large cracks in the next. The composite specimens deformed uniformly, with the CaS/HA preventing tissue damage and the bone preventing cracks in the CaS/HA from propagating through the specimen. In conclusion, using tomography with in situ loading, it was possible to show how CaS/HA can help prevent bone tissue damage before global failure.

Background: A prosthetic joint infection (PJI) is a devastating complication following total joint arthroplasties with poor prognosis. Identifying an accurate and prompt diagnostic method is particularly important for PJI. Recently, the diagnostic value of metagenomic next-generation sequencing (mNGS) in detecting PJI has attracted much attention, while the evidence of its accuracy is quite limited. Thus, this study aimed to evaluate the accuracy of mNGS for the diagnosis of PJI.

Methods: We summarized published studies to identify the potential diagnostic value of mNGS for PJI patients by searching online databases using keywords such as “prosthetic joint infection”, “PJI”, and “metagenomic sequencing”. Ten of 380 studies with 955 patients in total were included. The included studies provided sufficient data for the completion of 2-by-2 tables. We calculated the sensitivity, specificity, and area under the SROC curve (AUC) to evaluate mNGS for PJI diagnosis.

Results: We found that the pooled diagnostic sensitivity and specificity of mNGS for PJI were 0.93 (95% CI, 0.83 to 0.97) and 0.95 (95% CI, 0.92 to 0.97), respectively. Positive and negative likelihood ratios were 18.3 (95% CI, 10.9 to 30.6) and 0.07 (95% CI, 0.03 to 0.18), respectively. The area under the curve was 0.96 (95% CI, 0.93 to 0.97).

Conclusion: Metagenomic next-generation sequencing displays high accuracy in the diagnosis of PJI, especially for culture-negative cases

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.

Efficient systemic pharmacological treatment of solid tumors is hampered by inadequate tumor concentration of cytostatics necessitating development of smart local drug delivery systems. To overcome this, we demonstrate that doxorubicin (DOX), a cornerstone drug used for osteosarcoma treatment, shows reversible accretion to hydroxyapatite (HA) of both nano (nHA) and micro (mHA) size. nHA particles functionalized with DOX get engulfed in the lysosome of osteosarcoma cells where the acidic microenvironment causes a disruption of the binding between DOX and HA. The released DOX then accumulates in the mitochondria causing cell starvation, reduced migration and apoptosis. The HA+DOX delivery system was also tested in-vivo on osteosarcoma bearing mice. Locally delivered DOX via the HA particles had a stronger tumor eradication effect compared to the controls as seen by PET-CT and immunohistochemical staining of proliferation and apoptosis markers. These results indicate that in addition to systemic chemotherapy, an adjuvant nHA could be used as a carrier for intracellular delivery of DOX for prevention of tumor recurrence after surgical resection in an osteosarcoma. Furthermore, we demonstrate that nHA particles are pivotal in this approach but a combination of nHA with mHA could increase the safety associated with particulate nanomaterials while maintaining similar therapeutic potential.

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

In this study, we aimed to assess the stratification ability of the Fracture and Mortality Risk Evaluation (FAME) index for reoperation, new fragility fracture, and mortality during one-year follow-up.

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 microbiology and the susceptibility patterns of infected total knee arthroplasties (TKAs) vary depending on demographic, local antimicrobial stewardship, and surgical factors. We wanted to compare the recent microbial profile and antimicrobial resistance pattern in revisions due to infections after primary TKAs in Sweden and Lithuania. Our hypothesis was that there is a difference in bacteriology and resistance pattern based on patient related, societal and local hospital factors as almost similar praxis have been applied for TKA surgery, short term systemic prophylaxis and routine use of local gentamicin containing bone cement.

Doxorubicin (DOX) is a cornerstone drug in the treatment of osteosarcoma. However, achieving sufficient concentration in the tumor tissue after systemic administration with few side effects has been a challenge. Even with the most advanced nanotechnology approaches, less than 5% of the total administered drug gets delivered to the target site. Alternatives to increase the local concentration of DOX within the tumor using improved drug delivery methods are needed. In this study, we evaluate a clinically approved calcium sulfate/hydroxyapatite (CaS/HA) carrier, both in-vitro and in-vivo, for local, sustained and controlled delivery of DOX to improve osteosarcoma treatment. In-vitro drug release studies indicated that nearly 28% and 36% of the loaded drug was released over a period of 4-weeks at physiological pH (7.4) and acidic pH (5), respectively. About 63% of the drug had been released after 4-weeks in-vivo. The efficacy of the released drug from the CaS/HA material was verified on two human osteosarcoma cell lines MG-63 and 143B. It was demonstrated that the released drug fractions functioned the same way as the free drug without impacting its efficacy. Finally, the carrier system with DOX was assessed using two clinically relevant human osteosarcoma xenograft models. Compared to no treatment or the clinical standard of care with systemic DOX administration, the delivery of DOX using a CaS/HA biomaterial could significantly hinder tumor progression by inhibiting angiogenesis and cell proliferation. Our results indicate that a clinically approved CaS/HA biomaterial containing cytostatics could potentially be used for the local treatment of osteosarcoma.

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

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 poten- tially 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.

Background

Prosthetic joint infection (PJI) is the most serious total joint (TJA) complication despite several aseptic and antiseptic preventive measures. There is no clear evidence or even consensus, whether antibiotic-loaded bone cement (ALBC) should be used, in addition to systemic short-term routine antibiotic prophylaxis, to reduce the risk of PJI in primary TJA. We aimed to analyze the efficacy of ALBC for prevention of PJI in patients undergoing primary TJA.

Methods

We searched systematically for randomized controlled trials (RCTs) in PubMed, Scopus, Embase, Web of Science and Cochrane library. Two reviewers independently screened potentially eligible studies according to predefined selection criteria and assessed the risk of bias using a modified version of the Cochrane risk of bias tool. PJI was prespecified as the primary outcome of interest. The meta-analyses were based on risk ratios using random-effects model per default. For the purpose of sensitivity, the corresponding fixed effects model odds ratios were calculated with the use of the Peto method as well. To evaluate a potential difference in effect sizes using different types (subgroups) of antibiotics used in bone cement, and at different follow-up periods, we performed stratified meta-analyses.

Results

Thirty-seven studies were eligible for the systematic review and qualitative synthesis, and 9 trials (6507 total joint arthroplasties) were included in this meta-analysis. Overall ALBC significantly reduced the risk of PJI following primary TJAs (RRs, 0.36; 95% CIs, 0.16 to 0.80; P = 0.01) with a moderate degree of inconsistency (I² = 47%). Based on stratified meta-analyses the use of gentamicin appeared to have a better effect (P = 0.0005) in the total hip arthroplasty. Pooled data of different antibiotics used in knee arthroplasties showed a significant association of cefuroxime (RRs, 0.08; 95% CIs, 0.01 to 0.63; P = 0.02). Further, ALBCs significantly reduced the PJI at one and two years of follow-up (P = 0.03 and P = 0.005 respectively).

Conclusions
The evidence suggests that ALBCs are effective in reducing the PJI following primary TJA; i.e. between 20 and 84% reduced risk. However, the clear limitations of the available trial evidence highlight the need for joint-specific confirmatory trials, that will need to be designed as cluster-randomized trials of clinics in countries with well-functioning arthroplasty registries.

Background

Methods

Results

Fracture nonunions are common in orthopedics and their treatment often involves multiple surgical interventions. The aim of this study was to fabricate and characterize a gelatin–nano-hydroxyapatite membrane (GM)-based bone bandage for controlled delivery of bio-active molecules; recombinant human bone morphogenic protein-2 (rhBMP-2) and zoledronic acid (ZA) to promote osteoinduction and prevent callus resorption, respectively. In vitro cell–material interaction experiments using MC3T3 cells seeded on the GM indicated good biocompatibility. rhBMP-2-functionalized GM promoted osteogenic differentiation of MC3T3 cells and the rhBMP-2 bio-activity thus remained, as indicated by increased levels of alkaline phosphatase compared to only GM. The GM released a small amount (1.1%) of rhBMP-2 in vitro over a period of 5 weeks, demonstrating a strong interaction of rhBMP-2 with the GM. In the first animal study, the GM specimens loaded with rhBMP-2 or with the combination of rhBMP-2 + ZA were placed in the abdominal muscle pouch of rats. In the GM + rhBMP-2 + ZA group, significantly higher bone volume (21.5 ± 5.9 vs 2.7 ± 1.0 mm³) and area (3.3 ± 2.3 vs 1.0 ± 0.4 mm²) of bone were observed compared to GM + rhBMP-2 after 4 weeks, as indicated by micro-computed tomography and histomorphometry, respectively. Finally, a nonunion model in rats was used to evaluate the efficacy of the GM bandage and bio-active molecules in healing of fracture nonunions. The GM functionalized with rhBMP-2 + ZA led to higher bone formation around the fracture (63.9 ± 19.0 vs 31.8 ± 3.7 mm³) and stronger fracture callus (110.8 ± 46.8 vs 45.6 ± 17.8 N) compared to the empty controls. However, the overall union rate was only marginally improved. The GM alone or combined with ZA did not aid in bone healing in this model. Thus, this study shows that controlled delivery of rhBMP-2 + ZA via the developed GM is a promising approach that could aid in earlier full load bearing in patients with nonunion.

Background and purpose — Targeted delivery of drugs is important to achieve efficient local concentrations and reduce systemic side effects. We hypothesized that locally implanted synthetic hydroxyapatite (HA) particles can act as a recruiting moiety for systemically administered drugs, leading to targeted drug accretion. Methods — Synthetic HA particles were implanted ectopically in a muscle pouch in rats, and the binding of systemically circulating drugs such as zoledronic acid (ZA), tetracycline and 18F-fluoride (18F) was studied. The local biological effect was verified in an implant integration model in rats, wherein a hollow implant was filled with synthetic HA particles and the animals were given systemic ZA, 2-weeks post-implantation. The effect of HA particle size on drug binding and the possibility of reloading HA particles were also evaluated in the muscle pouch. Results — The systemically administered biomolecules (ZA, tetracycline and 18F) all sought the HA moiety placed in the muscle pouch. Statistically significant higher peri-implant bone volume and peak force were observed in the implant containing HA particles compared with the empty implant. After a single injection of ZA at 2 weeks, micro HA particles showed a tendency to accumulate more 14C-zoledronic acid (14C-ZA) than nano-HA particles in the muscle pouch. HA particles could be reloaded when ZA was given again at 4 weeks, showing increased 14C-ZA accretion by 73% in microparticles and 77% in nanoparticles. Interpretation — We describe a novel method of systemic drug loading resulting in targeted accretion in locally implanted particulate HA, thereby biologically activating the material.

About half a million people in Sweden (5%) (10.5 million inhabitants) walk around with a bone or joint implant. This could be extrapolated to 50 million only in the Americas, Europe, and the Pacific. Orthopedic surgeons are running the largest workshop for repairing humans worldwide, and, in fact, one-fifth of all people older than 75 years in Sweden have an artificial joint. In the United States, 5–10% of all hip or knee implants are being revised during a lifetime (Malchau et al. 2018).

The environmental as well the financial aspects of recycling metals at revision or post-mortem has thus far not been prioritized on the orthopedic societies’ agendas. Close to 100,000 Swedes pass away annually, with 70% being cremated. To protect the environment, the Swedish Government changed its regulation in 2016, making it mandatory for the Swedish Church—which is responsible for all funerals—to arrange for recycling after cremations of all metal components including the coffin. Since 2016, all metal collected at cremation has been recycled (Figure), resulting in 60 tons of valuable metals (such as, for instance, titanium) at a net value of US$15 million. The recycling company, however, charges 20%, while 80% is plowed back to support societal projects handled by a large general inheritance fund (Hyckenberg 2019). If the Swedish figures were extrapolated to Europe and the United States alone, the total income from metal recycling at funeral would be around US$250 million annually (i.e., 700 million inhabitants, assuming 1% mortality per year, 50% cremation and if 20% had a metal device at death) (The Cremation Society 2019). In addition, recycling of extracted implants that are currently scrapped as dangerous goods should be possible if the logistics for collection could be handled properly. This could be done under the auspices of the national orthopedic societies. With an expected minimal cumulative revision rate of 5% leading to a revision arthroplasty, this recycling in Sweden should give a yearly recurring income close to US$500,000, which could be directed to orthopedic research. Given the ongoing “age quake” and an increasing cremation trend in the industrialized world, metal implant recycling—which has just started in Sweden—is likely to spread to other countries. Leading implant manufacturers are encouraged to start collaborating with the orthopedic societies, who should assume their environmental responsibility and facilitate recycling of metal implants both at revision and at post-mortem. This has to be done in a manner respectful to the deceased and his or her loved ones.

In cooperation with patient organizations, we suggest that orthopedic societies put forward a motion to governments that at least part of the income from such recycling be directed to musculoskeletal research in the respective countries.

Project Specific Publication list

Bone Regeneration

Tumor

Yang L. et al., Longitudinal in vivo biodistribution of nano and micro sized hydroxyapatite particles implanted in a bone defect. Frontiers in Bioengineering and Biotechnology 2022. https://doi.org/10.3389/fbioe.2022.10763
Yang Liu., Hydroxyapatite – A trojan horse in the delivery of apatite-binding cytostatics in bone cancer. Doctoral Thesis 2022. https://portal.research.lu.se/en/publications/hydroxyapatite-a-trojan-horse-in-the-delivery-of-apatite-binding-
Yang L.et al., Bone mineral: A trojan horse for bone cancers efficient mitochondria targeted delivery and tumor eradication with nano hydroxyapatite containing doxorubicin. Materials Today Bio https://doi.org/10.1016/j.mtbio.2022.100227
Yang L. et al., Sustained and controlled delivery of doxorubicin from an in-situ setting hydroxyapatite carrier for local treatment of osteosarcoma. Acta Biomaterialia 2021. https://www.sciencedirect.com/science/article/pii/S1742706121004475
Yang L. et al., Nano-/micro- sized hydroxyapatite moiety improves doxorubicin delivery and tumor eradication., 2021
Yang L. et al., Dual delivery of doxorubicin and zoledronic acid from an injectable calcium sulphate/hydroxyapatite carrier.ORS 2020 Annual Meeting. Phoenix, Arizona. February 8 – 11, 2020. Abstract number 551.
Yang L. et al., Sequential and sustained release of doxorubicin and paclitaxel from a calcium sulfate/hydroxyapatite carrier.27^th Annual meeting of European Orthopedic Research Society (EORS 2019). Maastricht, Netherlands

Bone Infection

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