A review of biomacromolecule-based 3D bioprinting strategies for structure-function integrated repair of skin tissues.

When skin is damaged or affected by diseases, it often undergoes irreversible scar formation, leading to aesthetic concerns and psychological distress for patients. In cases of extensive skin defects, the patient's life can be severely compromised. In recent years, 3D printing technology has emerged as a groundbreaking approach to skin tissue engineering, offering promising solutions to various skin-related conditions. 3D bioprinting technology enables the precise fabrication of structures by programming the spatial arrangement of cells within the skin tissue and subsequently printing skin replacements either in a 3D bioprinter or directly at the site of the defect. This study provides a comprehensive overview of various biopolymer-based inks, with a particular emphasis on chitosan (CS), starch, alginate, agarose, cellulose, and fibronectin, all of which are natural polymers belonging to the category of biomacromolecules. Additionally, it summarizes artificially synthesized polymers capable of enhancing the performance of these biomacromolecule-based bioinks, thereby composing hybrid biopolymer inks aimed at better application in skin tissue engineering endeavors. This review paper examines the recent advancements, characteristics, benefits, and limitations of biological 3D bioprinting techniques for skin tissue engineering. By utilizing bioinks containing seed cells, hydrogels with bioactive factors, and biomaterials, complex structures resembling natural skin can be accurately fabricated in a layer-by-layer manner. The importance of biological scaffolds in promoting skin wound healing and the role of 3D bioprinting in skin tissue regeneration processes is discussed. Additionally, this paper addresses the challenges and constraints associated with current 3D bioprinting technologies for skin tissue and presents future perspectives. These include advancements in bioink formulations, full-thickness skin bioprinting, vascularization strategies, and skin appendages bioprinting.

Towards optimized tissue regeneration: a new 3D printable bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate.

Introduction: Autologous platelet concentrate (APC) are pro-angiogenic and can promote wound healing and tissue repair, also in combination with other biomaterials. However, challenging defect situations remain demanding. 3D bioprinting of an APC based bioink encapsulated in a hydrogel could overcome this limitation with enhanced physio-mechanical interface, growth factor retention/secretion and defect-personalized shape to ultimately enhance regeneration. Methods: This study used extrusion-based bioprinting to create a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate. Chemico-physical testing exhibited an amorphous structure characterized by high shape fidelity. Cytotoxicity assay and incubation of human osteogenic sarcoma cells (SaOs2) exposed excellent biocompatibility. enzyme-linked immunosorbent assay analysis confirmed pro-angiogenic growth factor release of the printed constructs, and co-incubation with HUVECS displayed proper cell viability and proliferation. Chorioallantoic membrane (CAM) assay explored the pro-angiogenic potential of the prints in vivo. Detailed proteome and secretome analysis revealed a substantial amount and homologous presence of pro-angiogenic proteins in the 3D construct. Results: This study demonstrated a 3D bioprinting approach to fabricate a novel bioink of alginate/cellulose hydrogel loaded with thrombocyte concentrate with high shape fidelity, biocompatibility, and substantial pro-angiogenic properties. Conclusion: This approach may be suitable for challenging physiological and anatomical defect situations when translated into clinical use.

Recent advances in metal-organic frameworks for stimuli-responsive drug delivery.

After entering the human body, drugs for treating diseases, which are prone to delivery and release in an uncontrolled manner, are affected by various factors. Based on this, many researchers utilize various microenvironmental changes encountered during drug delivery to trigger drug release and have proposed stimuli-responsive drug delivery systems. In recent years, metal-organic frameworks (MOFs) have become promising stimuli-responsive agents to release the loaded therapeutic agents at the target site to achieve more precise drug delivery due to their high drug loading, excellent biocompatibility, and high stimuli-responsiveness. The MOF-based stimuli-responsive systems can respond to various stimuli under pathological conditions at the site of the lesion, releasing the loaded therapeutic agent in a controlled manner, and improving the accuracy and safety of drug delivery. Due to the changes in different physical and chemical factors in the pathological process of diseases, the construction of stimuli-responsive systems based on MOFs has become a new direction in drug delivery and controlled release. Based on the background of the rapidly increasing attention to MOFs applied in drug delivery, we aim to review various MOF-based stimuli-responsive drug delivery systems and their response mechanisms to various stimuli. In addition, the current challenges and future perspectives of MOF-based stimuli-responsive drug delivery systems are also discussed in this review.

Metal-organic framework-based advanced therapeutic tools for antimicrobial applications.

The escalating concern over conventional antibiotic resistance has emphasized the urgency in developing innovative antimicrobial agents. In recent times, metal-organic frameworks (MOFs) have garnered significant attention within the realm of antimicrobial research due to their multifaceted antimicrobial attributes, including the sustained release of intrinsic or exogenous antimicrobial components, chemodynamically catalyzed generation of reactive oxygen species (ROS), and formation of photogenerated ROS. This comprehensive review provides a thorough overview of the synthetic approaches employed in the production of MOF-based materials, elucidating their underlying antimicrobial mechanisms in depth. The focal point lies in elucidating the research advancements across various antimicrobial modalities, encompassing intrinsic component release system, extraneous component release system, auto-catalytical system, and energy conversion system. Additionally, the progress of MOF-based antimicrobial materials in addressing wound infections, osteomyelitis, and periodontitis is meticulously elucidated, culminating in a summary of the challenges and potential opportunities inherent within the realm of antimicrobial applications for MOF-based materials. STATEMENT OF SIGNIFICANCE: Growing concerns about conventional antibiotic resistance emphasized the need for alternative antimicrobial solutions. Metal-organic frameworks (MOFs) have gained significant attention in antimicrobial research due to their diverse attributes like sustained antimicrobial components release, catalytic generation of reactive oxygen species (ROS), and photogenerated ROS. This review covers MOF synthesis and their antimicrobial mechanisms. It explores advancements in intrinsic and extraneous component release, auto-catalysis, and energy conversion systems. The paper also discusses MOF-based materials' progress in addressing wound infections, osteomyelitis, and periodontitis, along with existing challenges and opportunities. Given the lack of related reviews, our findings hold promise for future MOF applications in antibacterial research, making it relevant to your journal's readership.

Measuring Bone Healing: Parameters and Scores in Comparison.

(1) Background: Bone healing is a complex process that can not be replicated in its entirety in vitro. Research on bone healing still requires the animal model. The critical size femur defect (CSFD) in rats is a well-established model for fractures in humans that exceed the self-healing potential. New therapeutic approaches can be tested here in vivo. Histological, biomechanical, and radiological parameters are usually collected and interpreted. However, it is not yet clear to what extent they correlate with each other and how necessary it is to record all parameters. (2) Methods: The basis for this study was data from three animal model studies evaluating bone healing. The µCT and histological (Movat pentachrome, osteocalcin) datasets/images were reevaluated and correlation analyses were then performed. Two image processing procedures were compared in the analysis of the image data. (3) Results: There was a significant correlation between the histologically determined bone fraction (Movat pentachrome staining) and bending stiffness. Bone fraction determined by osteocalcin showed no prognostic value. (4) Conclusions: The evaluation of the image datasets using ImageJ is sufficient and simpler than the combination of both programs. Determination of the bone fraction using Movat pentachrome staining allows conclusions to be drawn about the biomechanics of the bone. A standardized procedure with the ImageJ software is recommended for determining the bone proportion.

Design and fabrication of intracellular therapeutic cargo delivery systems based on nanomaterials: current status and future perspectives.

Intracellular cargo delivery, the introduction of small molecules, proteins, and nucleic acids into a specific targeted site in a biological system, is an important strategy for deciphering cell function, directing cell fate, and reprogramming cell behavior. With the advancement of nanotechnology, many researchers use nanoparticles (NPs) to break through biological barriers to achieving efficient targeted delivery in biological systems, bringing a new way to realize efficient targeted drug delivery in biological systems. With a similar size to many biomolecules, NPs possess excellent physical and chemical properties and a certain targeting ability after functional modification on the surface of NPs. Currently, intracellular cargo delivery based on NPs has emerged as an important strategy for genome editing regimens and cell therapy. Although researchers can successfully deliver NPs into biological systems, many of them are delivered very inefficiently and are not specifically targeted. Hence, the development of efficient, target-capable, and safe nanoscale drug delivery systems to deliver therapeutic substances to cells or organs is a major challenge today. In this review, on the basis of describing the research overview and classification of NPs, we focused on the current research status of intracellular cargo delivery based on NPs in biological systems, and discuss the current problems and challenges in the delivery process of NPs in biological systems.

The effect of losartan on the development of post-traumatic joint stiffness in a rat model.

Post-traumatic joint stiffness (PTJS) is accompanied by a multidimensional disturbance of joint architecture. Pharmacological approaches represent promising alternatives as the traumatic nature of current therapeutic standards may lead to PTJS' progression. Losartan is an auspicious candidate, as it has demonstrated an antifibrotic effect in other organs. Forty-eight Sprague Dawley rats were randomized into equally sized losartan or control groups. After a standardized knee trauma, the joint was immobilized for either 2 weeks (n = 16), 4 weeks (n = 16) or 4 weeks with re-mobilization for an additional 4 weeks (n = 16). Pharmacotherapy with losartan or placebo (30 mg/kg/day) was initiated on the day of trauma and continued for the entire course. Joint contracture was measured alongside histological and molecular biological assessments. There were no significant biomechanical changes in joint contracture over time, comparing short-term (2 weeks) with long-term losartan therapy (4 weeks). However, comparing the formation of PTJS with that of the control, there was a trend toward improvement of joint mobility of 10.5° (p 0.09) under the influence of losartan. During the re-mobilization phase, no significant effect of losartan on range of motion (ROM) was demonstrated. At a cellular level, losartan significantly reduced myofibroblast counts by up to 72 % (4 weeks, p ≤ 0.001) without effecting the capsular configuration. Differences in expression levels of profibrotic factors (TGF-ß, CTGF, Il-6) were most pronounced at week 4. The antifibrotic properties of losartan are not prominent enough to completely prevent the development of PTJS after severe joint injury.

A new 3D-printed polylactic acid-bioglass composite for bone tissue engineering induces angiogenesis in vitro and in ovo.

Large bone defects such as those that occur after trauma or resections due to cancer still are a challenge for surgeons. Main challenge in this area is to find a suitable alternative to the gold-standard therapy, which is highly risky, and a promising option is to use biomaterials manufactured by 3D printing. In former studies, we demonstrated that the combination of polylactic acid (PLA) and bioglass (BG) resulted in a stable 3D-printable material, and porous and finely structured scaffolds were printed. These scaffolds exhibited osteogenic and anti-inflammatory properties. This 3D-printed material fulfills most of the requirements described in the diamond concept of bone healing. However, the question remains as to whether it also meets the requirements concerning angiogenesis. Therefore, the aim of this study was to analyze the effects of the 3D-printed PLA-BG composite material on angiogenesis. In vitro analyses with human umbilical vein endothelial cells (HUVECs) showed a positive effect of increasing BG content on viability and gene expression of endothelial markers. This positive effect was confirmed by an enhanced vascular formation analyzed by Matrigel assay and chicken chorioallantoic membrane (CAM) assay. In this work, we demonstrated the angiogenic efficiency of a 3D-printed PLA-BG composite material. Recalling the osteogenic potential of this material demonstrated in former work, we manufactured a mechanically stable, 3D-printable, osteogenic and angiogenic material, which could be used for bone tissue engineering.

Integrated osteoimmunomodulatory strategies based on designing scaffold surface properties in bone regeneration.

Those who have used traditional biomaterials as bone substitutes have always regarded the immune response as an obstacle leading to implant failure. However, cumulative evidence revealed that blindly minimizing host immune reactions cannot induce successful bone regeneration. With the emergence of the new concept of osteoimmunology, the intimate mutual effects between the skeletal system and the immune system have been gradually recognized, promoting the innovation of biomaterials with osteoimmunomodulatory properties. By tuning the surface properties, biomaterials can precisely manipulate the osteoimmune environment favoring bone regeneration. In this review, we first reviewed the mutual effects between the skeletal system and the immune system to show the importance of immunomodulation on bone regeneration. Subsequently, we summarize the recent developments in surface modification strategies in terms of the surface physicochemical properties and surface coatings and explain how these modification strategies work.

Recent Advances in Decellularized Extracellular Matrix-Based Bioinks for 3D Bioprinting in Tissue Engineering.

In recent years, three-dimensional (3D) bioprinting has been widely utilized as a novel manufacturing technique by more and more researchers to construct various tissue substitutes with complex architectures and geometries. Different biomaterials, including natural and synthetic materials, have been manufactured into bioinks for tissue regeneration using 3D bioprinting. Among the natural biomaterials derived from various natural tissues or organs, the decellularized extracellular matrix (dECM) has a complex internal structure and a variety of bioactive factors that provide mechanistic, biophysical, and biochemical signals for tissue regeneration and remodeling. In recent years, more and more researchers have been developing the dECM as a novel bioink for the construction of tissue substitutes. Compared with other bioinks, the various ECM components in dECM-based bioink can regulate cellular functions, modulate the tissue regeneration process, and adjust tissue remodeling. Therefore, we conducted this review to discuss the current status of and perspectives on dECM-based bioinks for bioprinting in tissue engineering. In addition, the various bioprinting techniques and decellularization methods were also discussed in this study.

Oxygen-Releasing Hyaluronic Acid-Based Dispersion with Controlled Oxygen Delivery for Enhanced Periodontal Tissue Engineering.

Periodontitis is a chronic biofilm-associated inflammatory disease of the tooth-supporting tissues that causes tooth loss. It is strongly associated with anaerobic bacterial colonization and represents a substantial global health burden. Due to a local hypoxic environment, tissue regeneration is impaired. Oxygen therapy has shown promising results as a potential treatment of periodontitis, but so far, local oxygen delivery remains a key technical challenge. An oxygen (O2)-releasing hyaluronic acid (HA)-based dispersion with a controlled oxygen delivery was developed. Cell viability of primary human fibroblasts, osteoblasts, and HUVECs was demonstrated, and biocompatibility was tested using a chorioallantoic membrane assay (CAM assay). Suppression of anaerobic growth of Porphyromonas gingivalis was shown using the broth microdilution assay. In vitro assays showed that the O2-releasing HA was not cytotoxic towards human primary fibroblasts, osteoblasts, and HUVECs. In vivo, angiogenesis was enhanced in a CAM assay, although not to a statistically significant degree. Growth of P. gingivalis was inhibited by CaO2 concentrations higher than 256 mg/L. Taken together, the results of this study demonstrate the biocompatibility and selective antimicrobial activity against P. gingivalis for the developed O2-releasing HA-based dispersion and the potential of O2-releasing biomaterials for periodontal tissue regeneration.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Angiogenesis In Vitro and In Ovo.

Bone fracture healing is a multistep process, including early immunological reactions, osteogenesis, and as a key factor, angiogenesis. Molecules inducing osteogenesis as well as angiogenesis are rare, but hold promise to be employed in bone tissue engineering. It has been demonstrated that the bone sialoprotein (BSP) can induce bone formation when immobilized in collagen type I, but its effect on angiogenesis still has to be characterized in detail. Therefore, the aim of this study was to analyse the effects of BSP immobilized in a collagen type I gel on angiogenesis. First, in vitro analyses with endothelial cells (HUVECs) were performed detecting enhancing effects of BSP on proliferation and gene expression of endothelial markers. A spheroid model was employed confirming these results. Finally, the inducing impact of BSP-collagen on vascular density was proved in a yolk sac membrane assay. Our results demonstrate that BSP is capable of inducing angiogenesis and confirm that collagen type I is the optimal carrier for this protein. Taking into account former results, and literature showing that BSP also induces osteogenesis, one can hypothesize that BSP couples angiogenesis and osteogenesis, making it a promising molecule to be used in bone tissue regeneration.

pH-Dependent Release of Vancomycin from Modularly Assembled Collagen Laminates.

To prevent surgical site infections, antibiotics can be released from carriers made of biomaterials, such as collagen, that support the healing process and are slowly degraded in the body. In our labs we have developed collagen laminates that can be easily assembled and bonded on-site, according to medical needs. As shown previously, the asymmetric assembly leads to different release rates at the major faces of the laminate. Since the pH changes during the wound healing and infection, we further examined the effect of an acidic and alkaline pH, in comparison to pH 7.4 on the release of vancomycin from different collagen samples. For this purpose, we used an additively manufactured sample holder and quantified the release by HPLC. Our results show that the pH value does not have any influence on the total amount of released vancomycin (atelocollagen sponge pH 5.5: 71 ± 2%, pH 7.4: 68 ± 8%, pH 8.5: 74 ± 3%, bilayer laminate pH 5.5: 61 ± 6%, pH 7.4: 69 ± 4% and pH 8.5: 67 ± 3%) but on the time for half-maximal release. At an acidic pH of 5.5, the swelling of the atelocollagen sponge is largely increased, leading to a 2-3 h retarded release, compared to the physiological pH. No changes in swelling were observed at the basic pH and the compound release was 1-2 h delayed. These effects need to be considered when choosing the materials for the laminate assembly.

In vitro Evaluation of a 20% Bioglass-Containing 3D printable PLA Composite for Bone Tissue Engineering.

Three-dimensional (3D) printing is considered a key technology in the production of customized scaffolds for bone tissue engineering. In a previous work, we developed a 3D printable, osteoconductive, hierarchical organized scaffold system. The scaffold material should be osteoinductive. Polylactic acid (PLA) (polymer)/Bioglass (BG) (mineral/ion source) composite materials are promising. Previous studies of PLA/BG composites never exceed BG fractions of 10%, as increase of bioactive BG component negatively affects the printability of the composite material. Here, we test a novel, 3D printable PLA/BG composite with BG fractions up to 20% for its biological activity in vitro. PLA/BG filaments suitable for microstructure 3D printing were spun and the effect of different BG contents (5%, 10%, and 20%) in this material on mesenchymal stem cell (MSC) activity was tested in vitro. Our results showed that all tested composites are biocompatible. MSC cell adherence and metabolic activity increase with increasing BG content. The presence of BG component in scaffold has only slight effect on osteogenic gene expression, but it has significant suppressive effect on the expression of inflammatory genes in MSC. In addition, the material did not provoke any significant inflammatory response in whole-blood stimulation assay. The results show that by increasing the BG content, the bioactivity can be further enhanced.

Bone Sialoprotein Immobilized in Collagen Type I Enhances Bone Regeneration In vitro and In vivo.

The use of bioactive molecules is a promising approach to enhance the bone healing properties of biomaterials. The aim of this study was to define the role of bone sialoprotein (BSP) immobilized in collagen type I in various settings. In vitro studies with human primary osteoblasts in mono- or in co-culture with endothelial cells demonstrated a slightly increased gene expression of osteogenic markers as well as an increased proliferation rate in osteoblasts after application of BSP immobilized in collagen type I. Two critical size bone defect models were used to analyze bone regeneration. BSP incorporated in collagen type I increased bone regeneration only marginally at one concentration in a calvarial defect model. To induce the mechanical stability, three-dimensional printing was used to produce a stable porous cylinder of polylactide. The cylinder was filled with collagen type I and immobilized BSP and implanted into a femoral defect of critical size in rats. This hybrid material was able to significantly induce bone regeneration. Our study clearly shows the osteogenic effect of BSP when combined with collagen type I as carrier and thereby offers various approaches and options for its use as bioactive molecule in bone substitute materials.

3D-Printed PLA-Bioglass Scaffolds with Controllable Calcium Release and MSC Adhesion for Bone Tissue Engineering.

Large bone defects are commonly treated by replacement with auto- and allografts, which have substantial drawbacks including limited supply, donor site morbidity, and possible tissue rejection. This study aimed to improve bone defect treatment using a custom-made filament for tissue engineering scaffolds. The filament consists of biodegradable polylactide acid (PLA) and a varying amount (up to 20%) of osteoconductive S53P4 bioglass. By employing an innovative, additive manufacturing technique, scaffolds with optimized physico-mechanical and biological properties were produced. The scaffolds feature adjustable macro- and microporosity (200-2000 µm) with adaptable mechanical properties (83-135 MPa). Additionally, controllable calcium release kinetics (0-0.25 nMol/µL after 24 h), tunable mesenchymal stem cell (MSC) adhesion potential (after 24 h by a factor of 14), and proliferation (after 168 h by a factor of 18) were attained. Microgrooves resulting from the 3D-printing process on the surface act as a nucleus for cell aggregation, thus being a potential cell niche for spheroid formation or possible cell guidance. The scaffold design with its adjustable biomechanics and the bioglass with its antimicrobial properties are of particular importance for the preclinical translation of the results. This study comprehensibly demonstrates the potential of a 3D-printed bioglass composite scaffold for the treatment of critical-sized bone defects.

Pleiotropic Long-Term Effects of Atorvastatin on Posttraumatic Joint Contracture in a Rat Model.

The antifibrotic effect of atorvastatin has already been demonstrated in several organ systems. In the present study, a rat model was used to investigate the effect of atorvastatin on posttraumatic joint contracture. Forty-eight Sprague Dawley rats were equally randomized into an atorvastatin group and a control group. After initial joint trauma, knee joints were immobilized for intervals of 2 weeks (n = 16) or 4 weeks (n = 16) or immobilized for 4 weeks with subsequent remobilization for another 4 weeks (n = 16). Starting from the day of surgery, animals received either atorvastatin or placebo daily. After euthanasia at week 2, 4 or 8, joint contracture was determined, histological examinations were performed, and gene expression was assessed. The results suggest that the joint contracture was primarily arthrogenic. Atorvastatin failed to significantly affect contracture formation and showed a reduction in myofibroblast numbers to 98 ± 58 (control: 319 ± 113, p < 0.01) and a reduction in joint capsule collagen to 60 ± 8% (control: 73 ± 9%, p < 0.05) at week 2. Gene expression of α-smooth muscle actin (α-SMA), collagen type I, transforming growth factor ß1 (TGF-ß1) and interleukin-6 (IL-6) was not significantly affected by atorvastatin. Atorvastatin decreases myofibroblast number and collagen deposition but does not result in an improvement in joint mobility.

Mechanical characterization of rose bengal and green light crosslinked collagen scaffolds for regenerative medicine.

Collagen is one of the most important biomaterials for tissue engineering approaches. Despite its excellent biocompatibility, it shows the non-negligible disadvantage of poor mechanical stability. Photochemical crosslinking with rose bengal and green light (RGX) is an appropriate method to improve this property. The development of collagen laminates is helpful for further adjustment of the mechanical properties as well as the controlled release of incorporated substances. In this study, we investigate the impact of crosslinking and layering of two different collagen scaffolds on the swelling behavior and mechanical behavior in micro tensile tests to obtain information on its wearing comfort (stiffness, strength and ductility). The mechanical stability of the collagen material after degradation due to cell contact is examined using thickness measurements. There is no linear increase or decrease due to layering homologous laminates. Unexpectedly, a decrease in elongation at break, Young's modulus and ultimate tensile strength are measured when the untreated monolayer is compared to the crosslinked one. Furthermore, we can detect a connection between stability and cell proliferation. The results show that with variation in number and type of layers, collagen scaffolds with tailored mechanical properties can be produced. Such a multi-layered structure enables the release of biomolecules into inner or outer layers for biomedical applications.

Progress of Phototherapy Applications in the Treatment of Bone Cancer.

Bone cancer including primary bone cancer and metastatic bone cancer, remains a challenge claiming millions of lives and affecting the life quality of survivors. Conventional treatments of bone cancer include wide surgical resection, radiotherapy, and chemotherapy. However, some bone cancer cells may remain or recur in the local area after resection, some are highly resistant to chemotherapy, and some are insensitive to radiotherapy. Phototherapy (PT) including photodynamic therapy (PDT) and photothermal therapy (PTT), is a clinically approved, minimally invasive, and highly selective treatment, and has been widely reported for cancer therapy. Under the irradiation of light of a specific wavelength, the photosensitizer (PS) in PDT can cause the increase of intracellular ROS and the photothermal agent (PTA) in PTT can induce photothermal conversion, leading to the tumoricidal effects. In this review, the progress of PT applications in the treatment of bone cancer has been outlined and summarized, and some envisioned challenges and future perspectives have been mentioned. This review provides the current state of the art regarding PDT and PTT in bone cancer and inspiration for future studies on PT.

An Additively Manufactured Sample Holder to Measure the Controlled Release of Vancomycin from Collagen Laminates.

The controlled release of antibiotics prevents the spread of pathogens and thereby improves healing processes in regenerative medicine. However, high concentrations may interfere with healing processes. It is therefore advantageous to use biodegradable materials for a controlled release. In particular, multilayer materials enable differential release at different surfaces. For this purpose, collagen sheets of different properties can be bonded by photochemical crosslinking. Here, we present the development and application of an easily accessible, additively manufactured sample holder to study the controlled release of vancomycin from modularly assembled collagen laminates in two directions. As proof-of-concept, we show that laminates of collagen sheets covalently linked by rose bengal and green light crosslinking (RGX) can be tightly inserted into the device without leakage from the upper to lower cavity. We used this sample holder to detect the release of vancomycin from symmetrically and asymmetrically loaded two-layer and three-layer collagen laminates into the upper and lower cavity of the sample holder. We show that these collagen laminates are characterized by a collagen type-dependent vancomycin release, enabling the control of antibiotic release profiles as well as the direction of antibiotic release.