Predicting in vitro human mesenchymal stromal cell expansion based on individual donor characteristics using machine learning.

BACKGROUND: Human mesenchymal stromal cells (hMSCs) have become attractive candidates for advanced medical cell-based therapies. An in vitro expansion step is routinely used to reach the required clinical quantities. However, this is influenced by many variables including donor characteristics, such as age and gender, and culture conditions, such as cell seeding density and available culture surface area. Computational modeling in general and machine learning in particular could play a significant role in deciphering the relationship between the individual donor characteristics and their growth dynamics. METHODS: In this study, hMSCs obtained from 174 male and female donors, between 3 and 64 years of age with passage numbers ranging from 2 to 27, were studied. We applied a Random Forests (RF) technique to model the cell expansion procedure by predicting the population doubling time (PDT) for each passage, taking into account individual donor-related characteristics. RESULTS: Using the RF model, the mean absolute error between model predictions and experimental results for the PDT in passage 1 to 4 is significantly lower compared with the errors obtained with theoretical estimates or historical data. Moreover, statistical analysis indicate that the PD and PDT in different age categories are significantly different, especially in the youngest group (younger than 10 years of age) compared with the other age groups. DISCUSSION: In summary, we introduce a predictive computational model describing in vitro cell expansion dynamics based on individual donor characteristics, an approach that could greatly assist toward automation of a cell expansion culture process.

Neurofibromatosis type 1-related pseudarthrosis: Beyond the pseudarthrosis site.

Neurofibromatosis type 1 (NF1) is an autosomal dominant disorder affecting approximately 1 in 2,000 newborns. Up to 5% of NF1 patients suffer from pseudarthrosis of a long bone (NF1-PA). Current treatments are often unsatisfactory, potentially leading to amputation. To gain more insight into the pathogenesis we cultured cells from PA tissue and normal-appearing periosteum of the affected bone for NF1 mutation analysis. PA cells were available from 13 individuals with NF1. Biallelic NF1 inactivation was identified in all investigated PA cells obtained during the first surgery. Three of five cases sampled during a later intervention showed biallelic NF1 inactivation. Also, in three individuals, we examined periosteum-derived cells from normal-appearing periosteum proximal and distal to the PA. We identified the same biallelic NF1 inactivation in the periosteal cells outside the PA region. These results indicate that NF1 inactivation is required but not sufficient for the development of NF1-PA. We observed that late-onset NF1-PA occurs and is not always preceded by congenital bowing. Furthermore, the failure to identify biallelic inactivation in two of five later interventions and one reintervention with a known somatic mutation indicates that NF1-PA can persist after the removal of most NF1 negative cells.

Developmental engineering of living implants for deep osteochondral joint surface defects.

INTRODUCTION: The repair of deep osteochondral joint surface defects represents a significant unmet clinical need. Importantly, untreated lesions lead to a high rate of osteoarthritis. The current strategies to repair these defects include osteochondral autograft transplantation or "sandwich" strategies combining bone autografts with autologous chondrocyte implantation, with poorly documented long-term outcomes. In this study, we first investigated the capacity of juvenile osteochondral grafts (OCGs) to repair osteochondral defects in skeletally mature rats. With this regenerative model in view, we produced a new biological, bilayered and scaffold-free Tissue Engineered construct (bTEC) for the repair of a deep osteochondral defect of the rat knee. METHODS: Cylindrical OCGs were excised from the femoral intercondylar groove of the knee of skeletally immature rats (5 weeks) and transplanted into osteochondral defects created in skeletally mature rats (11 weeks). To create bTECs, micromasses (µMasses) of human periosteum-derived progenitor cells (hPDCs) and human articular chondrocytes (hACs) were produced in vitro using previously optimized chemically defined medium formulations containing growth and differentiation factors including bone morphogenetic proteins. These two µMass types were subsequently implanted as bilayered constructs into osteochondral defects in nude rats. At 4 and 16 weeks after surgery, the knees were collected and processed for subsequent 3D imaging analysis and histological evaluation. Micro-computed tomography (µCT), H&E, and Safranin O staining were used to evaluate the degree and quality of tissue repair. RESULTS: The osteochondral unit of the knee joint in 5 weeks old rats exhibits an immature phenotype, displaying active subchondral bone formation through endochondral ossification and the absence of a tidemark. When transplanted into skeletally mature animals, the immature OCGs resumed their maturation process, i.e., formed new subchondral bone, established the tidemark, and maintained their Safranin O-positive hyaline cartilage at 16 weeks after transplantation. The bTECs (hPDCs + hACs) could partially recapitulate the biology as seen with the immature OCGs, including the formation of the joint surface architecture with typical zonation, ranging from non-mineralized hyaline cartilage in the superficial layers to a progressively mineralized matrix at the interface with a new subchondral bone plate. CONCLUSIONS: Cell-based TE constructs mimicking immature OCGs and displaying a hierarchically organized structure comprising of different tissue forming units seem an attractive strategy to treat deep osteochondral defects of the knee.

A cell-based combination product for the repair of large bone defects.

Regenerative cell-based implants using periosteum-derived stem cells were developed for the treatment of large 3 cm fresh and 4.5 centimeter biological compromised bone gaps in a tibial sheep model and compared with an acellular ceramic-collagen void filler. It was hypothesized that the latter is insufficient to heal large skeletal defects due to reduced endogenous biological potency. To this purpose a comparison was made between the ceramic dicalciumphosphate scaffold (CopiOs®) as such, the same ceramic coated with clinical grade Bone Morphogenetic Protein 2 and 6 (BMP) only or a BMP coated cell-seeded combination product. These implants were evaluated in 2 sheep models, a fresh 3 cm critical size tibial defect and a 4.5 cm biologically exhausted tibial defect. For the groups in which growth factors were applied, BMP-6 was chosen at a dose of 344 µg for 3 cm and 1.500 µg or 3.800 µg for 4.5 cm defects. An additional group in the 4.5 cm defect was tested using BMP-2 in a dose of 1.500 µg. For all the cell based implants autologous periosteum-derived cells were used which were cultured in monolayer during 6 weeks. For the fresh defect 408 million cells and for the biologically exhausted tibial defect 612 million cells were drop-seeded on the BMP coated scaffolds. Bone healing was studied during 16 weeks postimplantation, using standard radiographs. While fresh defects responded to all treatments, regardless the use of cells, the biologically hampered defects responded in half of the cases and only if the BMP-cell combination product was used, supporting the concept that cell-based therapies may become attractive in treating defects with a compromised biological status.

The Bone-Forming Properties of Periosteum-Derived Cells Differ Between Harvest Sites.

The development of alternatives for autologous bone grafts is a major focus of bone tissue engineering. To produce living bone-forming implants, skeletal stem and progenitor cells (SSPCs) are envisioned as key ingredients. SSPCs can be obtained from different tissues including bone marrow, adipose tissue, dental pulp, and periosteum. Human periosteum-derived cells (hPDCs) exhibit progenitor cell characteristics and have well-documented in vivo bone formation potency. Here, we have characterized and compared hPDCs derived from tibia with craniofacial hPDCs, from maxilla and mandible, respectively, each representing a potential source for cell-based tissue engineered implants for craniofacial applications. Maxilla and mandible-derived hPDCs display similar growth curves as tibial hPDCs, with equal trilineage differentiation potential toward chondrogenic, osteogenic, and adipogenic cells. These craniofacial hPDCs are positive for SSPC-markers CD73, CD164, and Podoplanin (PDPN), and negative for CD146, hematopoietic and endothelial lineage markers. Bulk RNA-sequencing identified genes that are differentially expressed between the three sources of hPDC. In particular, differential expression was found for genes of the HOX and DLX family, for SOX9 and genes involved in skeletal system development. The in vivo bone formation, 8 weeks after ectopic implantation in nude mice, was observed in constructs seeded with tibial and mandibular hPDCs. Taken together, we provide evidence that hPDCs show different profiles and properties according to their anatomical origin, and that craniofacial hPDCs are potential sources for cell-based bone tissue engineering strategies. The mandible-derived hPDCs display - both in vitro and in vivo - chondrogenic and osteogenic differentiation potential, which supports their future testing for use in craniofacial bone regeneration applications.

Bone augmentation with autologous periosteal cells and two different calcium phosphate scaffolds under an occlusive titanium barrier: an experimental study in rabbits.

BACKGROUND: This study used a tissue-engineering approach, which combined autologous periosteal cells with a scaffold material, to promote bone augmentation under an occlusive titanium barrier that was placed on the skull of rabbits. Because the cell-matrix interaction is of key importance in tissue engineering, two different calcium phosphate-based scaffolds were seeded with autologous periosteal cells. One scaffold contained hydroxyapatite, tricalcium phosphate, and collagen; the other scaffold was a beta-tricalcium phosphate structure. METHODS: The experiment involved 38 rabbits divided into five groups: the two different scaffolds with and without cells and a blood clot only. Prior to implantation, autologous periosteal cells were harvested from the tibia by stripping the periosteum. Cells were cultured, and 1 day before the implantation approximately 20 million cells were collected and seeded onto the scaffolds. Two preformed dome-shaped full titanium barriers were placed subperiosteally onto the frontal and parietal bones of each rabbit. Before placement of the barriers, the different scaffolds, seeded with or without cells, were put on top of the skull. As a negative control, autologous blood was injected into the barriers. Histologic evaluation and histomorphometric analysis were performed after 12 weeks of undisturbed bone growth. Measurements involved the amounts of newly formed tissue and of new bone distinguishing between trabecular bone and osteoid. RESULTS: No significant differences were found between the four treatment groups (scaffolds with or without cells). However, the amount of new bone tissue found underneath the titanium barriers with scaffolds was significantly higher (P <0.04) than with a blood clot only. CONCLUSION: A better understanding of the mode of action is required to optimize tissue-engineering procedures before entering clinical applications.

Deciphering the combined effect of bone morphogenetic protein 6 and calcium phosphate on bone formation capacity of periosteum derived cells-based tissue engineering constructs.

Cell based combination products with growth factors on optimal carriers represent a promising tissue engineering strategy to treat large bone defects. In this concept, bone morphogenetic protein (BMP) and calcium phosphate (CaP)-based scaffolds can act as potent components of the constructs to steer stem cell specification, differentiation and initiate subsequent in vivo bone formation. However, limited insight into BMP dosage and the cross-talk between BMP and CaP materials, hampers the optimization of in vivo bone formation and subsequent clinical translation. Herein, we combined human periosteum derived progenitor cells with different doses of BMP6 and with three types of clinical grade CaP-scaffolds (ChronOs®, ReproBone™, & CopiOs®). Comprehensive cellular and molecular analysis was performed based on in vitro cell metabolic activity and signaling pathway activation, as well as in vivo ectopic bone forming capacity after 2 weeks and 5 weeks in nude mice. Our data showed that cells seeded on CaP scaffolds with an intermediate Ca2+ release rate combined with low or medium dosage of BMP6 demonstrated a robust new bone formation after 5 weeks, which was contributed by both donor and host cells. This phenomenon might be due to the delicate balance between Ca2+ and BMP pathways, allowing an appropriate activation of the canonical BMP signaling pathway that is required for in vivo bone formation. For high BMP6 dosage, we found that the BMP6 dosage overrides the effect of the Ca2+ release rate and this appeared to be a dominant factor for ectopic bone formation. Taken together, this study illustrates the importance of matching BMP dosage and CaP properties to allow an appropriate activation of canonical BMP signaling that is crucial for in vivo bone formation. It also provides insightful knowledge with regard to clinical translation of cell-based constructs for bone regeneration. STATEMENT OF SIGNIFICANCE: The combination of bone morphogenetic proteins (BMP) and calcium phosphate (CaP)-based biomaterials with mesenchymal stromal cells represents a promising therapeutic strategy to treat large bone defects, an unmet medical need. However, there is limited insight into the optimization of these combination products, which hampers subsequent successful clinical translation. Our data reveal a delicate balance between Ca2+ and BMP pathways, allowing an appropriate activation of canonical BMP signaling required for in vivo bone formation. Our findings illustrate the importance of matching BMP dosage and CaP properties in the development of cell-based constructs for bone regeneration.

Warning About the Use of Critical-Size Defects for the Translational Study of Bone Repair: Analysis of a Sheep Tibial Model.

The repair of large long bone defects requires complex surgical procedures as the bone loss cannot simply be replaced by autologous grafts due to an insufficient bone stock of the human body. Tissue engineering strategies and the use of Advanced Therapy Medicinal Products (ATMPs) for these reconstructions remain a considerable challenge, in particular since robust outcomes in well-defined large animal models are lacking. To be suitable as a model for treatment of human sized bone defects, we developed a large animal model in both skeletally immature and mature sheep and made close observations on the spontaneous healing of defects. We warn for the spontaneous repair of large defects in immature animals, which can mask the (in)effectiveness of ATMP therapies, and propose the use of large 4.5 cm defects that are pretreated with a polymethylmethacrylate (PMMA) spacer in skeletally mature animals.

Patients' experience of pain and discomfort during instrumentation in the diagnosis and non-surgical treatment of periodontitis.

BACKGROUND: The present multicenter study aimed to survey the patients' experiences in relation to the diagnosis and non-surgical treatment of periodontitis according to current treatment routine. METHODS: Patients (N = 268) treated for plaque-related periodontitis were enrolled in two groups: group 1 received primary probing of pocket depths (PD 1) and/or primary scaling and root planing (SRP 1); group 2 consisted of patients who were scheduled in the different centers for recall PD/scaling/subgingival instrumentation. Data collection included a full periodontal status, anesthesia employed, procedure time, and patient self-completed questionnaires to assess their previous and current experience of pain and discomfort during anesthesia and periodontal treatment. RESULTS: Ninety percent of the patients received infiltration anesthesia during primary SRP, compared to only 2% during recall scaling/instrumentation and none during PD. Many patients (40% in SRP group 1 and 52% in the recall scaling/instrumentation group) were most bothered by the scaling procedure, while others (35% in the primary SRP patients) by the anesthetic injection. Painful experiences during previous primary PD and present SRP were highly correlated (r = 0.6). CONCLUSIONS: From these results, it is evident that subgingival instrumentation causes pain and discomfort, an aspect that should be considered in periodontal therapy. Unfortunately, the use of local infiltration anesthesia to reduce pain is in itself a cause of discomfort. Attractive alternatives for anesthetic applications are much needed.

Contrast-Enhanced Nanofocus X-Ray Computed Tomography Allows Virtual Three-Dimensional Histopathology and Morphometric Analysis of Osteoarthritis in Small Animal Models.

OBJECTIVE: One of the early hallmarks of osteoarthritis (OA) is a progressive degeneration of the articular cartilage. Early diagnosis of OA-associated cartilage alterations would be beneficial for disease prevention and control, and for the development of disease-modifying treatments. However, early diagnosis is still hampered by a lack of quantifiable readouts in preclinical models. DESIGN: In this study, we have shown the potency of contrast-enhanced nanofocus x-ray computed tomography (CE-nanoCT) to be used for virtual 3-dimensional (3D) histopathology in established mouse models for OA, and we compared with standard histopathology. RESULTS: We showed the equivalence of CE-nanoCT images to histopathology for the modified Mankin scoring of the cartilage structure and quality. Additionally, a limited set of 3D cartilage characteristics measured by CE-nanoCT image analysis in a user-independent and semiautomatic manner, that is, average and maximum of the noncalcified cartilage thickness distribution and loss in glycosaminoglycans, was shown to be predictive for the cartilage quality and structure as can be evaluated by histopathological scoring through the use of an empirical model. CONCLUSIONS: We have shown that CE-nanoCT is a tool that allows virtual histopathology and 3D morphological quantification of multitissue systems, such as the chondro-osseous junction. It provides faster and more quantitative data on cartilage structure and quality compared with standard histopathology while eliminating user bias. CE-nanoCT thus should allow capturing subtle differences in cartilage characteristics, carefully mapping OA progression and, ultimately, asses the beneficial changes when testing a candidate disease-modifying treatment.

In Vivo Evaluation of Different Surgical Procedures for Autologous Chondrocyte Implantation.

OBJECTIVE: Autologous chondrocyte implantation (ACI) involves the application of a chondrocyte suspension into a membrane-sealed cartilage defect. Recently, "cell-seeded collagen matrix-supported" ACI has been developed wherein chondrocytes are seeded on a biomembrane. This study aimed at preclinically comparing 4 variant ACI techniques in a refined goat model: 2 traditional procedures, whereby the defect is sealed by a periosteal flap or collagen membrane, and 2 cell-seeding methods, with the collagen membrane either sutured or glued into the defect. DESIGN: The efficacy of the surgical techniques was evaluated in an acute critical size chondral defect in the medial condyle of 32 skeletally mature goats, randomly assigned to 1 of the 4 aforementioned treatment groups. After 10 weeks in vivo, the quality of the repair was graded histologically by 2 independent, blinded readers using the "modified O'Driscoll" score. RESULTS: The cell-seeding procedure whereby the membrane is sutured into the defect has a similar structural repair capacity than traditional ACI techniques. However, when the cell-seeded membrane was glued into the defect, the outcome appeared inferior. CONCLUSION: These findings indicate that optimizing the goat model and the postoperative recovery does allow preclinical evaluation of ACI-based cartilage implants in a load-bearing setting. This preclinical observation provides support to the clinical utilization of the sutured membrane-seeded (ACI-CS) technique, provided sutures, but not fibrin sealants, are used to fix the cell-seeded membrane in the defect bed.

Histomorphometry and micro-computed tomography of bone augmentation under a titanium membrane.

OBJECTIVES: Bone augmentation underneath an occlusive titanium membrane is evaluated in most cases by means of serial histological sections and histomorphometry. Micro-computed tomography (micro-CT) is a less invasive and dynamic technique to measure bone volume in animals of a size that fits into the gantry. The aim of the present study was to evaluate whether the latter approach could match histomorphometry to assess bone augmentation under a titanium membrane. MATERIAL AND METHODS: Pre-formed titanium cups were placed on the skull of 16 rabbits. Bone formation underneath the cups was allowed to occur for 12 weeks. The amount of bone volume assessed by micro-CT was expressed as a numerical unit. One unit volume corresponds to 0.043 mm3. The measurements reveal the volume of bone-like tissue under the membrane, with the same density as that of the original rabbit skull bone. Histological sections were cut along the same plane as the one used for the micro-CT images. The total bone surface was assessed by a digital image system in double-stained undecalcified histological sections and related to the maximum available surface of the titanium cups, which was on average 1366 mm2. RESULTS: The amount of total bone surface found under the titanium membrane varied between 40 and 163 mm2. Measured by micro-CT, the bone detected ranged from 3.7 to 396 numerical units. A highly significant (P<0.001) correlation was found between the total bone volume measured in conventional serial histological sections and by the micro-CT technique (r2=0.72). CONCLUSIONS: The total bone volume measured underneath a membrane using the micro-CT when compared with histological sections remained within a 16% error. This is because of the scattering effect of the metallic membrane and the impossibility to distinguish newly formed bone from the original skull bone on the micro-CT images.