During the last decades, the combination of cellular and molecular biology and mechanical engineering, has stimulated the development of new multidisciplinary fields in biomedical sciences, such as tissue engineering and regenerative medicine. The primary goal of these approaches is to provide an alternative to organ and tissue transplantation, by creating viable substitutes for damaged or diseased organs and tissues. Tissue healing is a complex process that involves a cascade of cellular and molecular events, that are mostly shared by the different tissues of the body. Interestingly, the tissue repair process initiates immediately after a traumatic injury and is mediated and controlled by a wide range of cytokines, proteins and growth factors released from platelets upon activation. Consequently, many growth factors have being considered as therapeutic molecules for the repair or regeneration of a wide range of tissues. Although their role has been only partially elucidated, the potential benefit of most growth factors has been demonstrated. In the last few years, the development of platelet-rich preparations has revolutionized the field of regenerative medicine, due to the repair capacities of the platelet-released growth factors, that stimulate and accelerate both soft and hard tissue healing and regeneration. Today, platelet concentrates are used in a wide range of disciplines such as dentistry, oral surgery, orthopedics, sport medicine, dermatology, and plastic and reconstructive surgery. Currently, different techniques to obtain these hemocomponents are available, each leading to preparations that may differ in platelet concentration, leukocyte and fibrin content. Among them, there is Plasma Rich in Growth Factors (PRGF), a preparation rich in platelets, easily obtained from a small volume of patient’s blood, which, being autologous, does not promote any immune reaction and infectious disease transmission. Additionally, leukocytes are excluded from PRGF, so as to avoid any pro-inflammatory effects. Despite the growing craze for platelet concentrates technology, the scientific literature reported controversial results regarding the beneficial effect of these preparations on tissue healing, especially for bone regeneration. Considering all this information, the aim of our research was to developed a study model in order to gain more information about the biological effect of a platelet concentrate on both soft and hard tissue regeneration. For this purpose, human adipose-derived stem cells (hASCs), human osteoblasts (hObs) and human dermal fibroblasts (hDFs) cultured in the presence of PRGF, were studied. Usually, cells used for human therapy are expanded in fetal bovine serum (FBS), but this way carries the risk of potential immunogenic residual bovine proteins exposure and possible contamination with infectious agents, likely generating immune responses in patients. Therefore, in this study, FBS was completely substituted by the platelet concentrate for the cells culture. Interestingly, PRGF never affected cell viability and, after a short adaptation period, both hASCs and hDFs grown in the presence of PRGF, increased their proliferation rate compared to standard culture condition, while hObs growth was evident only without any cell’s detachment. Furthermore, neither donor nor cell population effect on both cell viability and proliferation was observed. Regarding osteo-differentiation, alkaline phoshatase (ALP) activity was induced in both hASCs and hObs, with a peak at day 7 and 14, respectively. In parallel, in order to elucidate the clinical benefit of platelet concentrates in hard tissue repair, the combination of a bone graft substitute with PRGF in enhancing a bone defect regeneration performed in a preclinical animal model, was also investigated. In particular, bilateral circular critical lesions were created in the proximal tibia of six New Zealand rabbits, for a total of 12 defects, filled with the bone substitute alone or in association with PRGF. Untreated defects were also included as control. No side-effects have been observed during the 8 weeks follow-up. Both histological and histomorphometric analysis showed a non-significant difference between treatment groups in terms of bone density, even if the addition of PRGF to the bone substitute seemed to induce a higher percentage of newly formed bone. In conclusion, this in vitro and in vivo study provided the evidence of the beneficial effect of platelet concentrates in both soft and hard tissue regeneration, thus supporting the application of this technology in clinical regenerative therapies.

PLATELET CONCENTRATE AND TISSUE REGENERATION: DOES IT WORK? A PRE-CLINICAL STUDY / C. Ceci ; tutor: M. Del Fabbro ; direttore della scuola di dottorato: R. L. Weinstein. - : . DIPARTIMENTO DI SCIENZE BIOMEDICHE, CHIRURGICHE ED ODONTOIATRICHE, 2015 Nov 19. ((28. ciclo, Anno Accademico 2015. [10.13130/c-ceci_phd2015-11-19].

PLATELET CONCENTRATE AND TISSUE REGENERATION: DOES IT WORK? A PRE-CLINICAL STUDY

C. Ceci
2015-11-19

Abstract

During the last decades, the combination of cellular and molecular biology and mechanical engineering, has stimulated the development of new multidisciplinary fields in biomedical sciences, such as tissue engineering and regenerative medicine. The primary goal of these approaches is to provide an alternative to organ and tissue transplantation, by creating viable substitutes for damaged or diseased organs and tissues. Tissue healing is a complex process that involves a cascade of cellular and molecular events, that are mostly shared by the different tissues of the body. Interestingly, the tissue repair process initiates immediately after a traumatic injury and is mediated and controlled by a wide range of cytokines, proteins and growth factors released from platelets upon activation. Consequently, many growth factors have being considered as therapeutic molecules for the repair or regeneration of a wide range of tissues. Although their role has been only partially elucidated, the potential benefit of most growth factors has been demonstrated. In the last few years, the development of platelet-rich preparations has revolutionized the field of regenerative medicine, due to the repair capacities of the platelet-released growth factors, that stimulate and accelerate both soft and hard tissue healing and regeneration. Today, platelet concentrates are used in a wide range of disciplines such as dentistry, oral surgery, orthopedics, sport medicine, dermatology, and plastic and reconstructive surgery. Currently, different techniques to obtain these hemocomponents are available, each leading to preparations that may differ in platelet concentration, leukocyte and fibrin content. Among them, there is Plasma Rich in Growth Factors (PRGF), a preparation rich in platelets, easily obtained from a small volume of patient’s blood, which, being autologous, does not promote any immune reaction and infectious disease transmission. Additionally, leukocytes are excluded from PRGF, so as to avoid any pro-inflammatory effects. Despite the growing craze for platelet concentrates technology, the scientific literature reported controversial results regarding the beneficial effect of these preparations on tissue healing, especially for bone regeneration. Considering all this information, the aim of our research was to developed a study model in order to gain more information about the biological effect of a platelet concentrate on both soft and hard tissue regeneration. For this purpose, human adipose-derived stem cells (hASCs), human osteoblasts (hObs) and human dermal fibroblasts (hDFs) cultured in the presence of PRGF, were studied. Usually, cells used for human therapy are expanded in fetal bovine serum (FBS), but this way carries the risk of potential immunogenic residual bovine proteins exposure and possible contamination with infectious agents, likely generating immune responses in patients. Therefore, in this study, FBS was completely substituted by the platelet concentrate for the cells culture. Interestingly, PRGF never affected cell viability and, after a short adaptation period, both hASCs and hDFs grown in the presence of PRGF, increased their proliferation rate compared to standard culture condition, while hObs growth was evident only without any cell’s detachment. Furthermore, neither donor nor cell population effect on both cell viability and proliferation was observed. Regarding osteo-differentiation, alkaline phoshatase (ALP) activity was induced in both hASCs and hObs, with a peak at day 7 and 14, respectively. In parallel, in order to elucidate the clinical benefit of platelet concentrates in hard tissue repair, the combination of a bone graft substitute with PRGF in enhancing a bone defect regeneration performed in a preclinical animal model, was also investigated. In particular, bilateral circular critical lesions were created in the proximal tibia of six New Zealand rabbits, for a total of 12 defects, filled with the bone substitute alone or in association with PRGF. Untreated defects were also included as control. No side-effects have been observed during the 8 weeks follow-up. Both histological and histomorphometric analysis showed a non-significant difference between treatment groups in terms of bone density, even if the addition of PRGF to the bone substitute seemed to induce a higher percentage of newly formed bone. In conclusion, this in vitro and in vivo study provided the evidence of the beneficial effect of platelet concentrates in both soft and hard tissue regeneration, thus supporting the application of this technology in clinical regenerative therapies.
DEL FABBRO, MASSIMO
WEINSTEIN, ROBERTO LODOVICO
Settore MED/28 - Malattie Odontostomatologiche
PLATELET CONCENTRATE AND TISSUE REGENERATION: DOES IT WORK? A PRE-CLINICAL STUDY / C. Ceci ; tutor: M. Del Fabbro ; direttore della scuola di dottorato: R. L. Weinstein. - : . DIPARTIMENTO DI SCIENZE BIOMEDICHE, CHIRURGICHE ED ODONTOIATRICHE, 2015 Nov 19. ((28. ciclo, Anno Accademico 2015. [10.13130/c-ceci_phd2015-11-19].
Doctoral Thesis
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/2434/330450
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