Dynamic co-evolution of transposable elements and the piRNA pathway in African cichlid fishes.

East African cichlid fishes have diversified in an explosive fashion, but the (epi)genetic basis of the phenotypic diversity of these fishes remains largely unknown. Although transposable elements (TEs) have been associated with phenotypic variation in cichlids, little is known about their transcriptional activity and epigenetic silencing. Here, we describe dynamic patterns of TE expression in African cichlid gonads and during early development. Orthology inference revealed an expansion of piwil1 genes in Lake Malawi cichlids, likely driven by PiggyBac TEs. The expanded piwil1 copies have signatures of positive selection and retain amino acid residues essential for catalytic activity. Furthermore, the gonads of African cichlids express a Piwi-interacting RNA (piRNA) pathway that target TEs. We define the genomic sites of piRNA production in African cichlids and find divergence in closely related species, in line with fast evolution of piRNA-producing loci. Our findings suggest dynamic co-evolution of TEs and host silencing pathways in the African cichlid radiations. We propose that this co-evolution has contributed to cichlid genomic diversity.

Lateral line system diversification during the early stages of ecological speciation in cichlid fish.

BACKGROUND: The mechanosensory lateral line system is an important sensory modality in fishes, informing multiple behaviours related to survival including finding food and navigating in dark environments. Given its ecological importance, we may expect lateral line morphology to be under disruptive selection early in the ecological speciation process. Here we quantify the lateral line system morphology of two ecomorphs of the cichlid fish Astatotilapia calliptera in crater Lake Masoko that have diverged from common ancestry within the past 1,000 years. RESULTS: Based on geometric morphometric analyses of CT scans, we show that the zooplanktivorous benthic ecomorph that dominates the deeper waters of the lake has large cranial lateral line canal pores, relative to those of the nearshore invertebrate-feeding littoral ecomorph found in the shallower waters. In contrast, fluorescence imaging revealed no evidence for divergence between ecomorphs in the number of either superficial or canal neuromasts. We illustrate the magnitude of the variation we observe in Lake Masoko A. calliptera in the context of the neighbouring Lake Malawi mega-radiation that comprises over 700 species. CONCLUSIONS: These results provide the first evidence of divergence in this often-overlooked sensory modality in the early stages of ecological speciation, suggesting that it may have a role in the broader adaptive radiation process.

Identification and management of low-risk isolated traumatic brain injury patients initially treated at a rural level IV trauma center.

STUDY OBJECTIVE: Our goal was to determine if low-risk, isolated mild traumatic brain injury (TBI) patients who were initially treated at a rural emergency department may have been safely managed without transfer to the tertiary referral trauma center. METHODS: This was a retrospective observational analysis of isolated mild TBI patients who were transferred from a rural Level IV Trauma Center to a regional Level I Trauma Center between 2018 and 2022. Patients were risk-stratified according to the modified Brain Injury Guidelines (mBIG). Data abstracted from the electronic medical record included patient presentation, management, and outcomes. RESULTS: 250 patients with isolated mild TBI were transferred out to the Level I Trauma Center. Fall was the most common mechanism of injury (69.2%). 28 patients (11.2%) were categorized as low-risk (mBIG1). No mBIG1 patients suffered a progression of neurological injury, had worsening of intracranial hemorrhage on repeat head CT, or required neurosurgical intervention. 12/28 (42.9%) of mBIG1 patients had a hospital length of stay of 2 days or less, typically for observation. Those with longer lengths of stay were due to medical complications, such as sepsis, or difficulty in arranging disposition. CONCLUSION: We propose that patients who meet mBIG1 criteria may be safely observed without transfer to a referral Level I Trauma Center. This would be of considerable benefit to patients, who would not need to leave their community, and would improve resource utilization in the region.

Predictors of direct oral anticoagulant concentrations in the trauma population.

Introduction: Direct oral anticoagulant (DOAC) use is becoming more prevalent in patients presenting after trauma. We sought to identify the prevalence and predictors of subtherapeutic and therapeutic DOAC concentrations and hypothesized that increased anti-Xa levels would correlate with increased risk of bleeding and other poor outcomes. Methods: A retrospective cohort study of all trauma patients on apixaban or rivaroxaban admitted to a level 1 trauma center between January 2015 and July 2021 was performed. Patients were excluded if they did not have a DOAC-specific anti-Xa level at presentation. Therapeutic levels were defined as an anti-Xa of 50 ng/mL to 250 ng/mL for rivaroxaban and 75 ng/mL to 250 ng/mL for apixaban. Linear regression was used to identify correlations between study variables and anti-Xa level, and binomial logistic regression was used to test the association of anti-Xa level with outcomes. Results: There were 364 trauma patients admitted during the study period who were documented to be on apixaban or rivaroxaban. Of these, 245 patients had anti-Xa levels measured at admission. The population was 53% woman, with median age of 78 years, and median Injury Severity Score of 5. In total, 39% of patients had therapeutic and 20% had supratherapeutic anti-Xa levels. Female sex, increased age, decreased height and weight, and lower estimated creatinine clearance were associated with higher anti-Xa levels at admission. There was no correlation between anti-Xa level and the need for transfusion or reversal agent administration, admission diagnosis of intracranial hemorrhage (ICH), progression of ICH, hospital length of stay, or mortality. Conclusions: Anti-Xa levels in trauma patients on DOACs vary widely; female patients who are older, smaller, and have decreased kidney function present with higher DOAC-specific anti-Xa levels after trauma. We were unable to detect an association between anti-Xa levels and clinical outcomes. Level of evidence: III-Prognostic and Epidemiological.

Metabolomic biomarkers in autism: identification of complex dysregulations of cellular bioenergetics.

Autism Spectrum Disorder (ASD or autism) is a phenotypically and etiologically heterogeneous condition. Identifying biomarkers of clinically significant metabolic subtypes of autism could improve understanding of its underlying pathophysiology and potentially lead to more targeted interventions. We hypothesized that the application of metabolite-based biomarker techniques using decision thresholds derived from quantitative measurements could identify autism-associated subpopulations. Metabolomic profiling was carried out in a case-control study of 499 autistic and 209 typically developing (TYP) children, ages 18-48 months, enrolled in the Children's Autism Metabolome Project (CAMP; ClinicalTrials.gov Identifier: NCT02548442). Fifty-four metabolites, associated with amino acid, organic acid, acylcarnitine and purine metabolism as well as microbiome-associated metabolites, were quantified using liquid chromatography-tandem mass spectrometry. Using quantitative thresholds, the concentrations of 4 metabolites and 149 ratios of metabolites were identified as biomarkers, each identifying subpopulations of 4.5-11% of the CAMP autistic population. A subset of 42 biomarkers could identify CAMP autistic individuals with 72% sensitivity and 90% specificity. Many participants were identified by several metabolic biomarkers. Using hierarchical clustering, 30 clusters of biomarkers were created based on participants' biomarker profiles. Metabolic changes associated with the clusters suggest that altered regulation of cellular metabolism, especially of mitochondrial bioenergetics, were common metabolic phenotypes in this cohort of autistic participants. Autism severity and cognitive and developmental impairment were associated with increased lactate, many lactate containing ratios, and the number of biomarker clusters a participant displayed. These studies provide evidence that metabolic phenotyping is feasible and that defined autistic subgroups can lead to enhanced understanding of the underlying pathophysiology and potentially suggest pathways for targeted metabolic treatments.

Agarose Fluid Gels Formed by Shear Processing During Gelation for Suspended 3D Bioprinting.

The use of granular matrices to support parts during the bioprinting process was first reported by Bhattacharjee et al. in 2015, and since then, several approaches have been developed for the preparation and use of supporting gel beds in 3D bioprinting. This paper describes a process to manufacture microgel suspensions using agarose (known as fluid gels), wherein particle formation is governed by the application of shear during gelation. Such processing produces carefully defined microstructures, with subsequent material properties that impart distinct advantages as embedding print media, both chemically and mechanically. These include behaving as viscoelastic solid-like materials at zero shear, limiting long-range diffusion, and demonstrating the characteristic shear-thinning behavior of flocculated systems. On the removal of shear stress, however, fluid gels have the capacity to rapidly recover their elastic properties. This lack of hysteresis is directly linked to the defined microstructures previously alluded to; because of the processing, reactive, non-gelled polymer chains at the particle interface facilitate interparticle interactions-similar to a Velcro effect. This rapid recovery of elastic properties enables bioprinting high-resolution parts from low-viscosity biomaterials, as rapid reformation of the support bed traps the bioink in situ, maintaining its shape. Furthermore, an advantage of agarose fluid gels is the asymmetric gelling/melting transitions (gelation temperature of ~30 °C and melting temperature of ~90 °C). This thermal hysteresis of agarose makes it possible to print and culture the bioprinted part in situ without the supporting fluid gel melting. This protocol shows how to manufacture agarose fluid gels and demonstrates their use to support the production of a range of complex hydrogel parts within suspended-layer additive manufacture (SLAM).

Erratum: Publisher's Note: "A suspended layer additive manufacturing approach to the bioprinting of tri-layered skin equivalents" [APL Bioeng. 5, 046103 (2021)].

[This corrects the article DOI: 10.1063/5.0061361.].

Cross-Border Antibiotic Resistance Patterns in Burn Patients.

Background: Antimicrobial resistance (AMR) is a growing problem worldwide, with differences in regional resistance patterns partially driven by local variance in antibiotic stewardship. Trauma patients transferring from Mexico have more AMR than those injured in the United States; we hypothesized a similar pattern would be present for burn patients. Patients and Methods: The registry of an American Burn Association (ABA)-verified burn center was queried for all admissions for burn injury January 2015 through December 2019 with hospital length-of-stay (LOS) longer than seven days. Patients were divided into two groups based upon burn location: United States (USA) or Mexico (MEX). All bacterial infections were analyzed. Results: A total of 73 MEX and 826 USA patients were included. Patients had a similar mean age (40.4 years MEX vs. 42.2 USA) and gender distribution (69.6% male vs. 64.4%). The MEX patients had larger median percent total body surface area burned (%TBSA; 11.1% vs. 4.3%; p ≤ 0.001) and longer hospital LOS (18.0 vs. 13.0 days; p = 0.028). The MEX patients more often had respiratory infections (16.4% vs. 7.4%; p = 0.046), whereas rates of other infections were similar. The MEX patients had higher rates of any resistant organism (47.2% of organisms MEX vs. 28.1% USA; p = 0.013), and were more likely to have resistant infections on univariable analysis; however, on multivariable analysis country of burn was no longer significant. Conclusions: Antimicrobial resistance is more common in burn patients initially burned in Mexico than those burned in the United States, but location was not a predictor of resistance compared to other traditional burn-related factors. Continuing to monitor for AMR regardless of country of burn remains critical.

Monitoring the stability of heparin: NMR evidence for the rearrangement of sulfated iduronate in phosphate buffer.

Heparin, a major anticoagulant drug, comprises a complex mixture of motifs. Heparin is isolated from natural sources while being subjected to a variety of conditions but the detailed effects of these on heparin structure have not been studied in depth. Therefore, the result of exposing heparin to a range of buffered environments, ranging pH values from 7 to 12, and temperatures of 40, 60 and 80 °C were examined. There was no evidence of significant N-desulfation or 6-O-desulfation in glucosamine residues, nor of chain scission, however, stereochemical re-arrangement of α-L-iduronate 2-O-sulfate to α-L-galacturonate residues occurred in 0.1 M phosphate buffer at pH 12/80 °C. The results confirm the relative stability of heparin in environments like those during extraction and purification processes; on the other hand, the sensitivity of heparin to pH 12 in buffered solution at high temperature is highlighted, providing an important insight for heparin manufacturers.

One size does not fit all: Sex bias in pharmacologic venous thromboembolism prophylaxis.

BACKGROUND: The optimal enoxaparin dosing strategy to achieve venous thromboembolism (VTE) prophylaxis in trauma patients remains unclear. Current dosing guidelines often include weight, age, and renal function but still fail to achieve appropriate prophylactic anti-Xa levels in many patients. We hypothesized that additional patient factors influence anti-Xa response to enoxaparin in trauma patients. METHODS: This is a retrospective review of patients admitted to a Level 1 trauma center for ≥4 days from July 2015 to September 2020, who received enoxaparin VTE prophylaxis per protocol (50-59 kg, 30 mg/dose; 60-99 kg, 40 mg/dose; ≥100 kg, 50 mg/dose; all doses every 12 hours) and had an appropriately timed peak anti-Xa level. Multivariate regression was performed to identify independent predictors of prophylactic anti-Xa levels (0.2-0.4 IU/mL) upon first measurement. RESULTS: The cohort (N = 1,435) was 76.4% male, with a mean ± SD age of 49.9 ± 20.0 years and a mean ± SD weight of 82.5 ± 20.2 kg (males, 85.2 kg; females, 73.7 kg; p <0.001). Overall, 68.6% of patients (n = 984) had a prophylactic anti-Xa level on first assessment (69.6% of males, 65.1% of females). Males were more likely to have a subprophylactic level than females (22.1% vs. 8.0%, p <0.001), whereas females were more likely to have supraprophylactic levels than males (26.9% vs. 8.3%, p < 0.001). When controlling for creatinine clearance, anti-Xa level was independently associated with dose-to-weight ratio (odds ratio, 0.191 for 0.5 mg/kg; p < 0.001; confidence interval, 0.151-0.230) and female sex (odds ratio, 0.060; p < 0.001; confidence interval, 0.047-0.072). Weight and age were not significant when controlling for the other factors. CONCLUSION: Male patients have a decreased anti-Xa response to enoxaparin when compared with female patients, leading to a greater incidence of subprophylactic anti-Xa levels in male patients at all dose-to-weight ratios. To improve the accuracy of VTE chemoprophylaxis, sex should be considered as a variable in enoxaparin dosing models. LEVEL OF EVIDENCE: Therapeutic/Care Management; Level III.

Two closely related ureotelic fish species of the genus Alcolapia express different levels of ammonium transporters in gills.

Most fish excrete their nitrogenous waste across the gills as ammonia through the activity of the Rhesus glycoprotein ammonium transporters. In contrast, fish of the subgenus Alcolapia (Oreochromis) are the only vertebrates that survive the extreme conditions of the soda lakes of Natron and Magadi in East Africa and have evolved adaptations to the highly alkaline waters including the ability to excrete their nitrogenous waste as urea. Nevertheless, Alcolapia retain the Rhesus glycoprotein genes in their genomes and using two heterologous expression systems, we demonstrate that Alcolapia Rhbg is capable of moving ammonia. Comparing ammonia and urea excretion from two closely related Alcolapia species from the same aquarium, we found that while Alcolapia grahami remains fully ureotelic after many generations in lab conditions, Alcolapia alcalica excretes some of its nitrogenous waste as ammonia. Using in situ hybridisation, we demonstrate robust, localised gene expression of Rhbg, rhcg1 and rhcg2 in the gill tissue in both A. alcalica embryos and adults, similar to that in other ammoniotelic fish. In contrast, the expression of these genes in A. grahami gills is much lower than in A. alcalica, suggesting the rapid evolution of a molecular mechanism underlying the complete ureotelism of A. grahami.

Management of choledocholithiasis in the elderly: Same-admission cholecystectomy remains the standard of care.

BACKGROUND: Current guidelines recommend that patients with choledocholithiasis undergo same-admission cholecystectomy. The compliance with this guideline is poor in elderly patients. We hypothesized that elderly patients treated with endoscopic retrograde cholangiopancreatography (ERCP) alone would have higher complication and readmission rates than the patients treated with cholecystectomy. METHODS: The Nationwide Readmissions Database was queried for all patients aged ≥65 years with admission for choledocholithiasis January to June 2016. The patients were divided based on index treatment received: (1) no intervention; (2) ERCP alone; or (3) cholecystectomy. Multivariate analyses identified predictors of cholecystectomy during index admission and of readmissions. RESULTS: A total of 16,121 patients with choledocholithiasis were admitted; 38.4% underwent cholecystectomy, 37.6% endoscopic retrograde cholangiopancreatography alone, and 24.0% no intervention. The patients not receiving a cholecystectomy were more likely to be older, female, have a higher Elixhauser score, do-not-resuscitate status, and at a teaching hospital (all P < .001). Emergency readmissions for recurrent biliary disease were lowest in patients undoing a cholecystectomy (2.2% vs 9.2% endoscopic retrograde cholangiopancreatography and 12.4% no intervention, P < .001), as were readmissions for complications (3.6% vs 5.5% and 7.8%, P < .001). Cholecystectomy reduced rates of readmissions for recurrent disease (odds ratio 0.168, P < .001), for complications (odds ratio 0.540, P < .001), and death during readmission (odds ratio 0.503, P = .007); endoscopic retrograde cholangiopancreatography alone reduced only rates of readmissions. Age was not a predictor of readmission or death. CONCLUSION: Index admission cholecystectomy is associated with a lower risk of readmission for biliary disease or complications, as well as death during readmission, in elderly patients. Age alone is not predictive of outcomes; surgical intervention should be guided by clinical condition, comorbidities, and patient preference.

Oca2 targeting using CRISPR/Cas9 in the Malawi cichlid Astatotilapia calliptera.

Identifying genetic loci underlying trait variation provides insights into the mechanisms of diversification, but demonstrating causality and characterizing the role of genetic loci requires testing candidate gene function, often in non-model species. Here we establish CRISPR/Cas9 editing in Astatotilapia calliptera, a generalist cichlid of the remarkably diverse Lake Malawi radiation. By targeting the gene oca2 required for melanin synthesis in other vertebrate species, we show efficient editing and germline transmission. Gene edits include indels in the coding region, probably a result of non-homologous end joining, and a large deletion in the 3' untranslated region due to homology-directed repair. We find that oca2 knock-out A. calliptera lack melanin, which may be useful for developmental imaging in embryos and studying colour pattern formation in adults. As A. calliptera resembles the presumed generalist ancestor of the Lake Malawi cichlids radiation, establishing genome editing in this species will facilitate investigating speciation, adaptation and trait diversification in this textbook radiation.

Youth violence prevention can be enhanced by geospatial analysis of trauma registry data.

BACKGROUND: Geographic information systems (GIS) have been used to understand relationships between trauma mechanisms, locations, and social determinants for injury prevention. We hypothesized that GIS analysis of trauma center registry data for assault patients aged 14 years to 29 years with census tract data would identify geospatial and structural determinants of youth violence. METHODS: Admissions to a Level I trauma center from 2010 to 2019 were retrospectively reviewed to identify assaults in those 14 years to 29 years. Prisoners were excluded. Home and injury scene addresses were geocoded. Cluster analysis was performed with the Moran I test for spatial autocorrelation. Census tract comparisons were done using American Communities Survey (ACS) data by t-test and linear regression. RESULTS: There were 1,608 admissions, 1,517 (92.4%) had complete addresses and were included in the analysis. Mean age was 23 ± 3.8 years, mean ISS was 7.5 ± 6.2, there were 11 (0.7%) in-hospital deaths. Clusters in six areas of the trauma catchment were identified with a Moran I value of 0.24 ( Z score = 17.4, p < 0.001). Linear regression of American Communities Survey demographics showed predictors of assault were unemployment (odds ratio, 4.5; 95% confidence interval, 2.7-6.4; p < 0.001), Spanish spoken at home (odds ratio, 6.6; 95% confidence interval, 3.4-9.8; p < 0.001) and poverty level (odds ratio, 1.9; 95% confidence interval, 1.1-2.7; p < 0.001). Education level of less than high school diploma, single parent households and race were not significant predictors. CONCLUSION: GIS analysis of registry data can identify high-risk areas for youth violence and correlated social and structural determinants. Violence prevention efforts can be better targeted geographically and socioeconomically with better understanding of these risk factors. LEVEL OF EVIDENCE: Prognostic/Epidemiological; Level III.

A suspended layer additive manufacturing approach to the bioprinting of tri-layered skin equivalents.

Skin exhibits a complex structure consisting of three predominant layers (epidermis, dermis, and hypodermis). Extensive trauma may result in the loss of these structures and poor repair, in the longer term, forming scarred tissue and associated reduction in function. Although a number of skin replacements exist, there have been no solutions that recapitulate the chemical, mechanical, and biological roles that exist within native skin. This study reports the use of suspended layer additive manufacturing to produce a continuous tri-layered implant, which closely resembles human skin. Through careful control of the bioink composition, gradients (chemical and cellular) were formed throughout the printed construct. Culture of the model demonstrated that over 21 days, the cellular components played a key role in remodeling the supporting matrix into architectures comparable with those of healthy skin. Indeed, it has been demonstrated that even at seven days post-implantation, the integration of the implant had occurred, with mobilization of the adipose tissue from the surrounding tissue into the construct itself. As such, it is believed that these implants can facilitate healing, commencing from the fascia, up toward the skin surface-a mechanism recently shown to be key within deep wounds.

Driving under the influence: a multi-center evaluation of vehicular crashes in the era of cannabis legalization.

BACKGROUND: Eleven states have instituted laws allowing recreational cannabis use leading to growing public health concerns surrounding the effects of cannabis intoxication on driving safety. We hypothesized that after the 2016 legalization of cannabis in California, the use among vehicular injury patients would increase and be associated with increased injury severity. METHODS: San Diego County's five adult trauma center registries in were queried from January 2010 to June 2018 for motor vehicle or motorcycle crash patients with completed toxicology screens. Patients were stratified as toxicology negative (TOX-), positive for only THC (THC+), only blood alcohol >0.08% (ETOH+), THC+ETOH, or THC+ with any combination with methamphetamine or cocaine (M/C). County medical examiner data were reviewed to characterize THC use in those with deaths at the scene of injury. RESULTS: Of the 11,491 patients identified, there were 61.6% TOX-, 11.7% THC+, 13.7% ETOH+, 5.0% THC+ETOH, and 7.9% M/C. THC+ increased from 7.3% to 14.8% over the study period and peaked at 14.9% post-legalization in 2017. Compared with TOX- patients, THC+ patients were more likely to be male and younger. THC+ patients were also less likely to wear seatbelts (8.5% vs 14.3%, p<0.001) and had increased mean Injury Severity Score (8.4±9.4 vs 9.0±9.9, p<0.001) when compared with TOX- patients. There was no difference in in-hospital mortality between groups. From the medical examiner data of the 777 deaths on scene, 27% were THC+. DISCUSSION: THC+ toxicology screens in vehicular injury patients peaked after the 2016 legalization of cannabis. Public education on the risks of driving under the influence of cannabis should be a component of injury prevention initiatives. LEVEL OF EVIDENCE: III, Prognostic.

Alginate Hydrogels with Tuneable Properties.

Alginate is a material that has many biomedical applications due to its low toxicity and a variety of favourable physical properties. In particular, the ease in which hydrogels are formed from alginate and the variety of mechanical behaviours that can be imparted on the hydrogels, by understanding alginate chemistry and intuitive design, has made alginate the most widely investigated polysaccharide used for tissue engineering. This chapter provides an overview of alginate, from how the source and natural variations in composition can influence mechanical properties of alginate hydrogels, through to some innovative techniques used to modify and functionalise the hydrogels designed specifically for cell-based therapies. The main focus is on how these strategies of understanding and controlling the chemistry of alginates have resulted in the development of hydrogels that can be tuned to deliver the physical behaviours required for successful application. This will also highlight how research on the physicochemical properties has helped alginate evolve from a structural polysaccharide in brown seaweed into a highly tuneable, multifunctional, smart biomaterial, which is likely to find further biomedical applications in the future.

Hydrolytic Degradation of Heparin in Acidic Environments: Nuclear Magnetic Resonance Reveals Details of Selective Desulfation.

Heparin is a complex glycosaminoglycan, derived mainly from pig mucosa, used therapeutically for its anticoagulant activity. Yet, owing largely to the chain complexity, the progressive effects of environmental conditions on heparin structure have not been fully described. A systematic study of the influence of acidic hydrolysis on heparin chain length and substitution has therefore been conducted. Changes in the sulfation pattern, monitored via 2D NMR, revealed initial de-N-sulfation of the molecule (pH 1/ 40 °C) and unexpectedly identified the secondary sulfate of iduronate as more labile than the 6-O-sulfate of glucosamine residues under these conditions (pH 1/ 60 °C). Additionally, the loss of sulfate groups, rather than depolymerization, accounted for most of the reduction in molecular weight. This provides an alternative route to producing partially 2-O-de-sulfated heparin derivatives that avoids using conventional basic conditions and may be of value in the optimization of processes associated with the production of heparin pharmaceuticals.

Heparin Enriched-WPI Coating on Ti6Al4V Increases Hydrophilicity and Improves Proliferation and Differentiation of Human Bone Marrow Stromal Cells.

Titanium alloy (Ti6Al4V) is one of the most prominent biomaterials for bone contact because of its ability to bear mechanical loading and resist corrosion. The success of Ti6Al4V implants depends on bone formation on the implant surface. Hence, implant coatings which promote adhesion, proliferation and differentiation of bone-forming cells are desirable. One coating strategy is by adsorption of biomacromolecules. In this study, Ti6Al4V substrates produced by additive manufacturing (AM) were coated with whey protein isolate (WPI) fibrils, obtained at pH 2, and heparin or tinzaparin (a low molecular weight heparin LMWH) in order to improve the proliferation and differentiation of bone-forming cells. WPI fibrils proved to be an excellent support for the growth of human bone marrow stromal cells (hBMSC). Indeed, WPI fibrils were resistant to sterilization and were stable during storage. This WPI-heparin-enriched coating, especially the LMWH, enhanced the differentiation of hBMSC by increasing tissue non-specific alkaline phosphatase (TNAP) activity. Finally, the coating increased the hydrophilicity of the material. The results confirmed that WPI fibrils are an excellent biomaterial which can be used for biomedical coatings, as they are easily modifiable and resistant to heat treatments. Indeed, the already known positive effect on osteogenic integration of WPI-only coated substrates has been further enhanced by a simple adsorption procedure.