Final Report Summary - BONEMIMIC (3D tissue-engineered model of bone adaptation)
• Summary of the project
One of the major challenges today is to diminish risk of bone fractures. Ageing of populations worldwide will be responsible for a major increase of the incidence of osteoporosis and therefore increase the risk of a bone fracture. In order to facilitate the development of advanced therapeutics in osteoporosis, preventive or even cures, it is imperative that we have a complete understanding of the cellular and molecular mechanisms of bone physiology and pathology.
Bone is a dynamic tissue that suffers a continuous process of bone formation and bone resorption (namely bone adaptation). A coordinated communication of osteoblast (Ob) cells, involved in formation of bone, and osteoclast (Oc) cells, which are the cells responsible of bone degradation, is a fundamental requirement for a balanced bone remodeling. Mechanical loads are an important modulator of bone architecture and cell physiology playing an important role in bone tissue homeostasis. Ob/Oc co-cultures are attractive because they are one step closer to natural conditions and allow elucidation of some aspects of the complex interactions between bone-building and bone-resorbing cell. Bioreactors provide a powerful system for the study and comprehension of the bone tissue development because they have the ability to create an in vitro situation that is controllable and enables mimicking the in vivo environment of bone. While several in vitro studies focus on the use of Ob/Oc in co-culture (Ob and Oc cells cultured together) in two dimensional (2D) and in three-dimensional (3D) models for biomaterials testing, little is known about the cell communication in a co-culture system under mechanical stimulation. Present work focuses the establishment a co-culture based system able to mimic in vitro normal bone remodeling, with the purpose to identify, to characterize and develop of new bioactive components for their use in bone diseases treatments, such as osteoporosis. Compared to currently investigations, this project opens a new way to investigate into normal physiology of bone that closer mimic to what happens in human bones.
• Overview of the research approach, results and conclusions
The overall aim of this study has been to establish an Ob/Oc co-culture in a bioreactor-based system able to mimic in vitro bone adaptation. As described in the project description, the research falls into 3 objectives:
Objective 1. Setting of single cell culture conditions in a bioreactor system. The aim of this first objective was to establish the cell culture conditions to allow differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and human monocytes (hMn) cells towards the Ob or to the Oc lineage into a three dimensional (3D) scaffold either with or without mechanical stimulation.
The fellow has worked on the establishment of adequate cell culture operation conditions for the differentiation of hBMSCs towards the osteogenic lineage. Differentiation of hBMSCs to Ob cells is characterized by the formation of a mineralized extracellular matrix. Re-establishment of the cell culture conditions to allow hBMSCs differentiation towards the osteogenic lineage was required in order to allow the acquisition of enough mineral formation in vitro. Different fetal bovine serum (FBS) types were tested and compared. The selection of the FBS type showed to be crucial in the acquisition of a EBT. The new cell culture conditions have now been re-established and show to support the formation of mineralized extracellular matrix in a 3D environment in vitro, a critical objective for the success of this project. In addition, the fellow has been trained and established the operation conditions of a new spinner flask bioreactor. To provide a model that closely resemble the mechanical loads experienced by bone, we have further evaluated i) if new the cell culture conditions established supported hBMSCs differentiation and formation of mineralized matrix at two different types of fluid shear stress (spinner and perfusion) and ii) how the different mechanical loads are transmitted to and sensed by the cells has been evaluated by quantification of the gene. Although development of a mineralized matrix was achieved by all of the physical stimulations applied, differences in cell response, organization of the mineral deposition and bone mineral density among the bioreactor types were observed. To advance on the development of bone graft substitutes to accelerate repair of bone fractures, evaluation of how pore geometry of a 3D structure influence on how cell response under a mechanical stress has been further evaluated. Results showed that the structure on the 3D scaffolds affects on how cells sense and respond to mechanical application. In overall, the results displayed are of high importance for i) further applicability and feasibility of the proposed cell culture conditions with alternative bioreactor systems developed at host research group, ii) to provide information of tissue quality, iii) temporal monitoring of bone formation and iv) a better understanding on how cells respond to pore geometry and mechanical loads will help on the design of scaffolds for BTE.
Next, establishment of the protocols for isolation and purification of hMn cells from human buffy coats was addressed. Methods for hMn cell isolation and purification has been defined and are now well established. In addition, the selection of the appropriate cell culture media is specific for each cell type and is crucial to guarantee the success and functionality of a certain cell type. Due to that the culture media composition commonly used for hMn cells differ from the one used for the hBMSC cells, hMn cell survival and differentiation into Oc under that two media types was compared. Although temporal differences were observed in the formation of mature osteoclast cells, hMn were viable and could differentiate to Oc in culture media composition desired for our co-culture system. This result has represented a key factor towards the establishment of the co-cultured model. Results obtained proved that hMn cells can survive and differentiate into Oc cells, under growth factor supplementation, when cultured under the same media composition than the hBMSC cells.
Objective 2: Biological characterization of an Ob/Oc co-culture system. The aim of this objective is to define that conditions that allows a coordinated activity of both cell types.
The fellow has worked on the setting and the establishment of a cell 3D co-culture model to mimic the bone remodelling in vitro. The work carried out has focused on i) providing a 3D environment to the cells similar to natural bone ii) the establishment of a 3D co-culture of Ob and Oc under a mechanical stimulus. As described in the project description, the objective 2 was marked by a strong training element. To provide a structure that resemble the bone tissue structure and composition, methods for the formation of a biocompatible engineered bone tissue (EBT), characterized by mineralized tissue and the inexistence of precursor cells, has been successfully obtained. The biocompatibility of the EBT has been confirmed and showed to support cell adhesion and survival. Further, the cell survival and differentiation of hBMCS and hMn cultured on the EBT formed has been further evaluated. The influence of different regimes of culturing (temporality for hBMSC/hMn cell seeding and different modifications in media composition) on differentiation of hBMSC and hMn cultured to Ob or Oc cells has been compared under mechanical stimulation (spinner flask bioreactor). Results have been analyzed, by immunohistochemistry, quantification of the gene expression and by monitoring of the formation and degradation of the mineral tissue over the co-culture time. This objective has been characterized by the setting up of several new methods, which has been all needed for the final implementation on the co-culture model. This work will provide high value information about how the culture regime studied affect on the activity of both cell types to lead a coordinated activity that mimic the bone adaptation process that occurs in vivo.
Objective 3: Evaluation of the biological response of well-known therapeutic molecules in a co-culture system. The most suitable co-culture condition obtained from objective 2 will be selected for the testing of the expected effects of a drug clinically used for osteoporosis treatment.
To improve the quality of the research and the experimental conditions to support single and co-cell culture, more time than expected was needed to be invested on objective 1 and 2. In addition, this project was characterized by a strong training element. Together, this resulted in a change in the timeline proposed in the work plan and thus, the objective 3 has not been reached.
• Expected socio-economic impact:
This project integrates an innovative and advanced approach to investigate into normal physiology of bone and for development of new treatment of bone diseases. To comprehend tissue development and to develop optimal treatment is synonymous of welfare of society.
From the pharmaceutical-industrial view, a reliable model system facilitating the understanding the bone adaptation process that occurs in normal conditions, will be useful for initial testing of the drug safety and for the evaluation of their effectivity on bone treatment disease such as osteoporosis. It is expected that the drug screening of osteoactive molecules in the co-culture model established in an in-house designed bioreactor will further provide 3D monitoring of the effects of a drug (e.g. antiresorptive) on density of the mineralized matrix. Moreover, the proposed model would be highly beneficial for the pharmaceutical industry because it might enable high-throughput in vitro drug screening and reduce animal experiments. From the clinical perspective, the effects of a drug administered to a patient have to be well known and reproducible. Thus, the identification, development and characterization of the effects of potential drugs for bone disease in a reproducible controlled and automated system introduced by bioreactors and in a 3D system will facilitate the transferability of the results from pre-clinical to clinical stage.
Currently the last objective of this project has not been achieved and thus further research will be needed for the assessment of the potential of the model proposed.
One of the major challenges today is to diminish risk of bone fractures. Ageing of populations worldwide will be responsible for a major increase of the incidence of osteoporosis and therefore increase the risk of a bone fracture. In order to facilitate the development of advanced therapeutics in osteoporosis, preventive or even cures, it is imperative that we have a complete understanding of the cellular and molecular mechanisms of bone physiology and pathology.
Bone is a dynamic tissue that suffers a continuous process of bone formation and bone resorption (namely bone adaptation). A coordinated communication of osteoblast (Ob) cells, involved in formation of bone, and osteoclast (Oc) cells, which are the cells responsible of bone degradation, is a fundamental requirement for a balanced bone remodeling. Mechanical loads are an important modulator of bone architecture and cell physiology playing an important role in bone tissue homeostasis. Ob/Oc co-cultures are attractive because they are one step closer to natural conditions and allow elucidation of some aspects of the complex interactions between bone-building and bone-resorbing cell. Bioreactors provide a powerful system for the study and comprehension of the bone tissue development because they have the ability to create an in vitro situation that is controllable and enables mimicking the in vivo environment of bone. While several in vitro studies focus on the use of Ob/Oc in co-culture (Ob and Oc cells cultured together) in two dimensional (2D) and in three-dimensional (3D) models for biomaterials testing, little is known about the cell communication in a co-culture system under mechanical stimulation. Present work focuses the establishment a co-culture based system able to mimic in vitro normal bone remodeling, with the purpose to identify, to characterize and develop of new bioactive components for their use in bone diseases treatments, such as osteoporosis. Compared to currently investigations, this project opens a new way to investigate into normal physiology of bone that closer mimic to what happens in human bones.
• Overview of the research approach, results and conclusions
The overall aim of this study has been to establish an Ob/Oc co-culture in a bioreactor-based system able to mimic in vitro bone adaptation. As described in the project description, the research falls into 3 objectives:
Objective 1. Setting of single cell culture conditions in a bioreactor system. The aim of this first objective was to establish the cell culture conditions to allow differentiation of human bone marrow mesenchymal stem cells (hBMSCs) and human monocytes (hMn) cells towards the Ob or to the Oc lineage into a three dimensional (3D) scaffold either with or without mechanical stimulation.
The fellow has worked on the establishment of adequate cell culture operation conditions for the differentiation of hBMSCs towards the osteogenic lineage. Differentiation of hBMSCs to Ob cells is characterized by the formation of a mineralized extracellular matrix. Re-establishment of the cell culture conditions to allow hBMSCs differentiation towards the osteogenic lineage was required in order to allow the acquisition of enough mineral formation in vitro. Different fetal bovine serum (FBS) types were tested and compared. The selection of the FBS type showed to be crucial in the acquisition of a EBT. The new cell culture conditions have now been re-established and show to support the formation of mineralized extracellular matrix in a 3D environment in vitro, a critical objective for the success of this project. In addition, the fellow has been trained and established the operation conditions of a new spinner flask bioreactor. To provide a model that closely resemble the mechanical loads experienced by bone, we have further evaluated i) if new the cell culture conditions established supported hBMSCs differentiation and formation of mineralized matrix at two different types of fluid shear stress (spinner and perfusion) and ii) how the different mechanical loads are transmitted to and sensed by the cells has been evaluated by quantification of the gene. Although development of a mineralized matrix was achieved by all of the physical stimulations applied, differences in cell response, organization of the mineral deposition and bone mineral density among the bioreactor types were observed. To advance on the development of bone graft substitutes to accelerate repair of bone fractures, evaluation of how pore geometry of a 3D structure influence on how cell response under a mechanical stress has been further evaluated. Results showed that the structure on the 3D scaffolds affects on how cells sense and respond to mechanical application. In overall, the results displayed are of high importance for i) further applicability and feasibility of the proposed cell culture conditions with alternative bioreactor systems developed at host research group, ii) to provide information of tissue quality, iii) temporal monitoring of bone formation and iv) a better understanding on how cells respond to pore geometry and mechanical loads will help on the design of scaffolds for BTE.
Next, establishment of the protocols for isolation and purification of hMn cells from human buffy coats was addressed. Methods for hMn cell isolation and purification has been defined and are now well established. In addition, the selection of the appropriate cell culture media is specific for each cell type and is crucial to guarantee the success and functionality of a certain cell type. Due to that the culture media composition commonly used for hMn cells differ from the one used for the hBMSC cells, hMn cell survival and differentiation into Oc under that two media types was compared. Although temporal differences were observed in the formation of mature osteoclast cells, hMn were viable and could differentiate to Oc in culture media composition desired for our co-culture system. This result has represented a key factor towards the establishment of the co-cultured model. Results obtained proved that hMn cells can survive and differentiate into Oc cells, under growth factor supplementation, when cultured under the same media composition than the hBMSC cells.
Objective 2: Biological characterization of an Ob/Oc co-culture system. The aim of this objective is to define that conditions that allows a coordinated activity of both cell types.
The fellow has worked on the setting and the establishment of a cell 3D co-culture model to mimic the bone remodelling in vitro. The work carried out has focused on i) providing a 3D environment to the cells similar to natural bone ii) the establishment of a 3D co-culture of Ob and Oc under a mechanical stimulus. As described in the project description, the objective 2 was marked by a strong training element. To provide a structure that resemble the bone tissue structure and composition, methods for the formation of a biocompatible engineered bone tissue (EBT), characterized by mineralized tissue and the inexistence of precursor cells, has been successfully obtained. The biocompatibility of the EBT has been confirmed and showed to support cell adhesion and survival. Further, the cell survival and differentiation of hBMCS and hMn cultured on the EBT formed has been further evaluated. The influence of different regimes of culturing (temporality for hBMSC/hMn cell seeding and different modifications in media composition) on differentiation of hBMSC and hMn cultured to Ob or Oc cells has been compared under mechanical stimulation (spinner flask bioreactor). Results have been analyzed, by immunohistochemistry, quantification of the gene expression and by monitoring of the formation and degradation of the mineral tissue over the co-culture time. This objective has been characterized by the setting up of several new methods, which has been all needed for the final implementation on the co-culture model. This work will provide high value information about how the culture regime studied affect on the activity of both cell types to lead a coordinated activity that mimic the bone adaptation process that occurs in vivo.
Objective 3: Evaluation of the biological response of well-known therapeutic molecules in a co-culture system. The most suitable co-culture condition obtained from objective 2 will be selected for the testing of the expected effects of a drug clinically used for osteoporosis treatment.
To improve the quality of the research and the experimental conditions to support single and co-cell culture, more time than expected was needed to be invested on objective 1 and 2. In addition, this project was characterized by a strong training element. Together, this resulted in a change in the timeline proposed in the work plan and thus, the objective 3 has not been reached.
• Expected socio-economic impact:
This project integrates an innovative and advanced approach to investigate into normal physiology of bone and for development of new treatment of bone diseases. To comprehend tissue development and to develop optimal treatment is synonymous of welfare of society.
From the pharmaceutical-industrial view, a reliable model system facilitating the understanding the bone adaptation process that occurs in normal conditions, will be useful for initial testing of the drug safety and for the evaluation of their effectivity on bone treatment disease such as osteoporosis. It is expected that the drug screening of osteoactive molecules in the co-culture model established in an in-house designed bioreactor will further provide 3D monitoring of the effects of a drug (e.g. antiresorptive) on density of the mineralized matrix. Moreover, the proposed model would be highly beneficial for the pharmaceutical industry because it might enable high-throughput in vitro drug screening and reduce animal experiments. From the clinical perspective, the effects of a drug administered to a patient have to be well known and reproducible. Thus, the identification, development and characterization of the effects of potential drugs for bone disease in a reproducible controlled and automated system introduced by bioreactors and in a 3D system will facilitate the transferability of the results from pre-clinical to clinical stage.
Currently the last objective of this project has not been achieved and thus further research will be needed for the assessment of the potential of the model proposed.