A polymeric protein-loaded microsphere delivery system for use in bone tissue engineering

The bone graft procedure is the gold standard therapy for large bone defects and usually involves implanting autologous bone into the defect site. The need to find more patient-friendly alternatives to the bone graft procedure is a driving force behind recent advances in bone tissue engineering. Different synthetic and natural biomaterials are being investigated for their use in the repair of large bone defects. However, there is still a need for scaffold materials that are able to sinter in situ with the capability of delivering growth factors that promote the bone repair process with minimal invasion. In this study, our main aim was to develop a porous, protein-loaded microsphere delivery system with the capability of sintering in situ and with injectable potential. Poly (lactic-co-glycolic acid) (PLGA) was used for this formulation due to its biodegradability, biocompatibility, controllable mechanical properties and good processing capabilities. An optimised procedure for formulating porous and non-porous (protein-loaded) microspheres was established and the microspheres were extensively characterised. There was an inverse relationship between the molecular weight of the PLGA and both the protein release and compressive strength. An intermediate molecular weight (53 kDa) variety of PLGA was chosen for further work based on balancing the need for retaining sufficient compressive strength and a slower protein release profile. The burst release of protein was addressed by investigating various coatings. The combination of chitosan and PLGA to form composite PLGA/chitosan microspheres resulted in the desired reduction in the burst release. The protein-loaded and porous microspheres were combined as a paste and found to sinter at body temperature (37°C) into scaffolds. Whilst previous investigations have focused primarily on a single type of PLGA microsphere (with or without an additional biomaterial), in this study we combined both porous and protein-loaded microspheres into a single delivery system. Furthermore, successful sintering was confirmed when a suspension of the microspheres was injected through a 19 G needle. The biocompatibility and osteogenic potential of the scaffolds were investigated. The composite PLGA/chitosan scaffolds supported the growth of MG-63 osteosarcoma cells and a primary human stem cell line. Furthermore, the scaffolds also supported the osteogenesis of the stem cells, as demonstrated by the presence of the late protein marker of osteogenesis, osteocalcin, and positive von Kossa staining, which is indicative of mineralization. The composite PLGA/chitosan and porous microspheres combine both porosity and the ability to load and sustain the release of protein into one system. Moreover, their ability to sinter at body temperature when injected or applied as a paste demonstrates the dual functionality of the system. This represents a novel approach to delivering protein for tissue regeneration as presently, there has been no report of the combination of PLGA/chitosan microspheres with porous PLGA microspheres as a system that is able to sinter, both post-injection and when packed as a paste, at 37°C. Therefore, the formulation presented in this thesis shows potential for further in vitro and in vivo testing to determine its suitability for bone tissue engineering applications

A dual-application poly (DL-lactic-co-glycolic) acid (PLGA)-chitosan composite scaffold for potential use in bone tissue engineering

The development of patient-friendly alternatives to bone-graft procedures is the driving force for new frontiers in bone tissue engineering. Poly (DL-lactic-co-glycolic acid), (PLGA) and chitosan are well-studied and easy-to-process polymers from which scaffolds can be fabricated. In this study, a novel dual-application scaffold system was formulated from porous PLGA and protein-loaded PLGA/chitosan microspheres. Physicochemical and in vitro protein release attributes were established. The therapeutic relevance, cytocompatibility with primary human mesenchymal stem cells (hMSCs) and osteogenic properties were tested. There was a significant reduction in burst release from the composite PLGA/chitosan microspheres compared with PLGA alone. Scaffolds sintered from porous microspheres at 37°C were significantly stronger than the PLGA control, with compressive strengths of 0.846 ± 0.272 MPa and 0.406 ± 0.265 MPa, respectively (p < 0.05). The formulation also sintered at 37°C following injection through a needle, demonstrating its injectable potential. The scaffolds demonstrated cytocompatibility, with increased cell numbers observed over an 8-day study period. Von Kossa and immunostaining of the hMSC-scaffolds confirmed their osteogenic potential with the ability to sinter at 37°C in situ

Deprivation, essential and non-essential activities and SARS-CoV-2 infection following the lifting of national public health restrictions in England and Wales [version 1; peer review: awaiting peer review]

BACKGROUND: Individuals living in deprived areas in England and Wales undertook essential activities more frequently and experienced higher rates of SARS-CoV-2 infection than less deprived communities during periods of restrictions aimed at controlling the Alpha (B.1.1.7) variant. We aimed to understand whether these deprivation-related differences changed once restrictions were lifted. METHODS: Among 11,231 adult Virus Watch Community Cohort Study participants multivariable logistic regressions were used to estimate the relationships between deprivation and self-reported activities and deprivation and infection (self-reported lateral flow or PCR tests and linkage to National Testing data and Second Generation Surveillance System (SGSS)) between August – December 2021, following the lifting of national public health restrictions. RESULTS: Among 11,231 adult Virus Watch Community Cohort Study participants multivariable logistic regressions were used to estimate the relationships between deprivation and self-reported activities and deprivation and infection (self-reported lateral flow or PCR tests and linkage to National Testing data and Second Generation Surveillance System (SGSS)) between August – December 2021, following the lifting of national public health restrictions. CONCLUSIONS: The lack of association between deprivation and infection is likely due to the increased engagement in non-essential activities among the least deprived balancing the increased work-related exposure among the most deprived. The differences in activities highlight stark disparities in an individuals’ ability to choose how to limit infection exposure

Development and Validation of a Primary Care Electronic Health Record Phenotype to Study Migration and Health in the UK.

International migrants comprised 14% of the UK's population in 2020; however, their health is rarely studied at a population level using primary care electronic health records due to difficulties in their identification. We developed a migration phenotype using country of birth, visa status, non-English main/first language and non-UK-origin codes and applied it to the Clinical Practice Research Datalink (CPRD) GOLD database of 16,071,111 primary care patients between 1997 and 2018. We compared the completeness and representativeness of the identified migrant population to Office for National Statistics (ONS) country-of-birth and 2011 census data by year, age, sex, geographic region of birth and ethnicity. Between 1997 to 2018, 403,768 migrants (2.51% of the CPRD GOLD population) were identified: 178,749 (1.11%) had foreign-country-of-birth or visa -status codes, 216,731 (1.35%) non-English-main/first-language codes, and 8288 (0.05%) non-UK-origin codes. The cohort was similarly distributed versus ONS data by sex and region of birth. Migration recording improved over time and younger migrants were better represented than those aged ≥50. The validated phenotype identified a large migrant cohort for use in migration health research in CPRD GOLD to inform healthcare policy and practice. The under-recording of migration status in earlier years and older ages necessitates cautious interpretation of future studies in these groups

SARS-CoV-2 infections in migrants and the role of household overcrowding: a causal mediation analysis of Virus Watch data

BACKGROUND: Migrants are over-represented in SARS-CoV-2 infections globally; however, evidence is limited for migrants in England and Wales. Household overcrowding is a risk factor for SARS-CoV-2 infection, with migrants more likely to live in overcrowded households than UK-born individuals. We aimed to estimate the total effect of migration status on SARS-CoV-2 infection and to what extent household overcrowding mediated this effect. METHODS: We included a subcohort of individuals from the Virus Watch prospective cohort study during the second SARS-CoV-2 wave (1 September 2020-30 April 2021) who were aged ≥18 years, self-reported the number of rooms in their household and had no evidence of SARS-CoV-2 infection pre-September 2020. We estimated total, indirect and direct effects using Buis' logistic decomposition regression controlling for age, sex, ethnicity, clinical vulnerability, occupation, income and whether they lived with children. RESULTS: In total, 23 478 individuals were included. 9.07% (187/2062) of migrants had evidence of infection during the study period vs 6.27% (1342/21 416) of UK-born individuals. Migrants had 22% higher odds of infection during the second wave (total effect; OR 1.22, 95% CI 1.01 to 1.47). Household overcrowding accounted for approximately 36% (95% CI -4% to 77%) of these increased odds (indirect effect, OR 1.07, 95% CI 1.03 to 1.12; proportion accounted for: indirect effect on log odds scale/total effect on log odds scale=0.36). CONCLUSION: Migrants had higher odds of SARS-CoV-2 infection during the second wave compared with UK-born individuals and household overcrowding explained 36% of these increased odds. Policy interventions to reduce household overcrowding for migrants are needed as part of efforts to tackle health inequalities during the pandemic and beyond

A case-crossover study of the effect of vaccination on SARS-CoV-2 transmission relevant behaviours during a period of national lockdown in England and Wales.

BACKGROUND: Studies of COVID-19 vaccine effectiveness show increases in COVID-19 cases within 14 days of a first dose, potentially reflecting post-vaccination behaviour changes associated with SARS-CoV-2 transmission before vaccine protection. However, direct evidence for a relationship between vaccination and behaviour is lacking. We aimed to examine the association between vaccination status and self-reported non-household contacts and non-essential activities during a national lockdown in England and Wales. METHODS: Participants (n = 1154) who had received the first dose of a COVID-19 vaccine reported non-household contacts and non-essential activities from February to March 2021 in monthly surveys during a national lockdown in England and Wales. We used a case-crossover study design and conditional logistic regression to examine the association between vaccination status (pre-vaccination vs 14 days post-vaccination) and self-reported contacts and activities within individuals. Stratified subgroup analyses examined potential effect heterogeneity by sociodemographic characteristics such as sex, household income or age group. RESULTS: 457/1154 (39.60 %) participants reported non-household contacts post-vaccination compared with 371/1154 (32.15 %) participants pre-vaccination. 100/1154 (8.67 %) participants reported use of non-essential shops or services post-vaccination compared with 74/1154 (6.41 %) participants pre-vaccination. Post-vaccination status was associated with increased odds of reporting non-household contacts (OR 1.65, 95 % CI 1.31-2.06, p < 0.001) and use of non-essential shops or services (OR 1.50, 95 % CI 1.03-2.17, p = 0.032). This effect varied between men and women and different age groups. CONCLUSION: Participants had higher odds of reporting non-household contacts and use of non-essential shops or services within 14 days of their first COVID-19 vaccine compared to pre-vaccination. Public health emphasis on maintaining protective behaviours during this post-vaccination time period when individuals have yet to develop full protection from vaccination could reduce risk of SARS-CoV-2 infection

Relative contribution of essential and non-essential activities to SARS-CoV-2 transmission following the lifting of public health restrictions in England and Wales

PURPOSE: We aimed to understand which non-household activities increased infection odds and contributed greatest to SARS-CoV-2 infections following the lifting of public health restrictions in England and Wales. PROCEDURES: We undertook multivariable logistic regressions assessing the contribution to infections of activities reported by adult Virus Watch Community Cohort Study participants. We calculated adjusted weighted population attributable fractions (aPAF) estimating which activity contributed greatest to infections. FINDINGS: Among 11 413 participants (493 infections), infection was associated with: leaving home for work (aOR 1.35 (1.11-1.64), aPAF 17%), public transport (aOR 1.27 (1.04-1.57), aPAF 12%), shopping once (aOR 1.83 (1.36-2.45)) vs. more than three times a week, indoor leisure (aOR 1.24 (1.02-1.51), aPAF 10%) and indoor hospitality (aOR 1.21 (0.98-1.48), aPAF 7%). We found no association for outdoor hospitality (1.14 (0.94-1.39), aPAF 5%) or outdoor leisure (1.14 (0.82-1.59), aPAF 1%). CONCLUSION: Essential activities (work and public transport) carried the greatest risk and were the dominant contributors to infections. Non-essential indoor activities (hospitality and leisure) increased risk but contributed less. Outdoor activities carried no statistical risk and contributed to fewer infections. As countries aim to 'live with COVID', mitigating transmission in essential and indoor venues becomes increasingly relevant

A polymeric protein-loaded microsphere delivery system for use in bone tissue engineering

The bone graft procedure is the gold standard therapy for large bone defects and usually involves implanting autologous bone into the defect site. The need to find more patient-friendly alternatives to the bone graft procedure is a driving force behind recent advances in bone tissue engineering. Different synthetic and natural biomaterials are being investigated for their use in the repair of large bone defects. However, there is still a need for scaffold materials that are able to sinter in situ with the capability of delivering growth factors that promote the bone repair process with minimal invasion. In this study, our main aim was to develop a porous, protein-loaded microsphere delivery system with the capability of sintering in situ and with injectable potential. Poly (lactic-co-glycolic acid) (PLGA) was used for this formulation due to its biodegradability, biocompatibility, controllable mechanical properties and good processing capabilities. An optimised procedure for formulating porous and non-porous (protein-loaded) microspheres was established and the microspheres were extensively characterised. There was an inverse relationship between the molecular weight of the PLGA and both the protein release and compressive strength. An intermediate molecular weight (53 kDa) variety of PLGA was chosen for further work based on balancing the need for retaining sufficient compressive strength and a slower protein release profile. The burst release of protein was addressed by investigating various coatings. The combination of chitosan and PLGA to form composite PLGA/chitosan microspheres resulted in the desired reduction in the burst release. The protein-loaded and porous microspheres were combined as a paste and found to sinter at body temperature (37°C) into scaffolds. Whilst previous investigations have focused primarily on a single type of PLGA microsphere (with or without an additional biomaterial), in this study we combined both porous and protein-loaded microspheres into a single delivery system. Furthermore, successful sintering was confirmed when a suspension of the microspheres was injected through a 19 G needle. The biocompatibility and osteogenic potential of the scaffolds were investigated. The composite PLGA/chitosan scaffolds supported the growth of MG-63 osteosarcoma cells and a primary human stem cell line. Furthermore, the scaffolds also supported the osteogenesis of the stem cells, as demonstrated by the presence of the late protein marker of osteogenesis, osteocalcin, and positive von Kossa staining, which is indicative of mineralization. The composite PLGA/chitosan and porous microspheres combine both porosity and the ability to load and sustain the release of protein into one system. Moreover, their ability to sinter at body temperature when injected or applied as a paste demonstrates the dual functionality of the system. This represents a novel approach to delivering protein for tissue regeneration as presently, there has been no report of the combination of PLGA/chitosan microspheres with porous PLGA microspheres as a system that is able to sinter, both post-injection and when packed as a paste, at 37°C. Therefore, the formulation presented in this thesis shows potential for further in vitro and in vivo testing to determine its suitability for bone tissue engineering applications